US20230140670A1 - Formulations - Google Patents

Formulations Download PDF

Info

Publication number
US20230140670A1
US20230140670A1 US16/651,911 US201816651911A US2023140670A1 US 20230140670 A1 US20230140670 A1 US 20230140670A1 US 201816651911 A US201816651911 A US 201816651911A US 2023140670 A1 US2023140670 A1 US 2023140670A1
Authority
US
United States
Prior art keywords
lipid
composition
mol
lnp
rna
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
US16/651,911
Other languages
English (en)
Inventor
Kristy M. Wood
Noah P. Gardner
Ruchi R. Shah
Stephen S. Scully
Ramsey Majzoub
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Intellia Therapeutics Inc
Original Assignee
Intellia Therapeutics Inc
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Intellia Therapeutics Inc filed Critical Intellia Therapeutics Inc
Priority to US16/651,911 priority Critical patent/US20230140670A1/en
Assigned to INTELLIA THERAPEUTICS, INC. reassignment INTELLIA THERAPEUTICS, INC. ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: GARDNER, NOAH P., MAJZOUB, Ramsey, SCULLY, STEPHEN S., SHAH, RUCHI R., WOOD, Kristy M.
Publication of US20230140670A1 publication Critical patent/US20230140670A1/en
Pending legal-status Critical Current

Links

Images

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
    • C12N15/88—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation using microencapsulation, e.g. using amphiphile liposome vesicle
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A61K31/7105—Natural ribonucleic acids, i.e. containing only riboses attached to adenine, guanine, cytosine or uracil and having 3'-5' phosphodiester links
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A61K48/0008—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'non-active' part of the composition delivered, e.g. wherein such 'non-active' part is not delivered simultaneously with the 'active' part of the composition
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0019—Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/10—Dispersions; Emulsions
    • A61K9/127—Synthetic bilayered vehicles, e.g. liposomes or liposomes with cholesterol as the only non-phosphatidyl surfactant
    • A61K9/1271—Non-conventional liposomes, e.g. PEGylated liposomes or liposomes coated or grafted with polymers
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/10—Dispersions; Emulsions
    • A61K9/127—Synthetic bilayered vehicles, e.g. liposomes or liposomes with cholesterol as the only non-phosphatidyl surfactant
    • A61K9/1271—Non-conventional liposomes, e.g. PEGylated liposomes or liposomes coated or grafted with polymers
    • A61K9/1272—Non-conventional liposomes, e.g. PEGylated liposomes or liposomes coated or grafted with polymers comprising non-phosphatidyl surfactants as bilayer-forming substances, e.g. cationic lipids or non-phosphatidyl liposomes coated or grafted with polymers
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
    • A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
    • A61K9/51—Nanocapsules; Nanoparticles
    • A61K9/5107—Excipients; Inactive ingredients
    • A61K9/5123—Organic compounds, e.g. fats, sugars
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
    • C12N9/22—Ribonucleases [RNase]; Deoxyribonucleases [DNase]
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/20—Type of nucleic acid involving clustered regularly interspaced short palindromic repeats [CRISPR]
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/31—Chemical structure of the backbone
    • C12N2310/315—Phosphorothioates
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/32—Chemical structure of the sugar
    • C12N2310/321—2'-O-R Modification
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/34—Spatial arrangement of the modifications
    • C12N2310/346—Spatial arrangement of the modifications having a combination of backbone and sugar modifications
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/35—Nature of the modification
    • C12N2310/352—Nature of the modification linked to the nucleic acid via a carbon atom
    • C12N2310/3521—Methyl
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2320/00—Applications; Uses
    • C12N2320/30—Special therapeutic applications
    • C12N2320/32—Special delivery means, e.g. tissue-specific
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2800/00—Nucleic acids vectors
    • C12N2800/80—Vectors containing sites for inducing double-stranded breaks, e.g. meganuclease restriction sites

Definitions

  • LNP compositions with improved properties for delivery of biologically active agents, in particular RNAs, mRNAs, and guide RNAs are provided herein.
  • the LNP compositions facilitate delivery of RNA agents across cell membranes, and in particular embodiments, they introduce components and compositions for gene editing into living cells.
  • Biologically active agents that are particularly difficult to deliver to cells include proteins, nucleic acid-based drugs, and derivatives thereof.
  • Compositions for delivery of promising gene editing technologies into cells, such as for delivery of CRISPR/Cas9 system components, are of particular interest.
  • CRISPR/Cas gene editing systems are active as ribonucleoprotein complexes in a cell.
  • An RNA-directed nuclease binds to and directs cleavage of a DNA sequence in the cell.
  • This site-specific nuclease activity facilitates gene editing through the cell's own natural processes.
  • the cell responds to double-stranded DNA breaks (DSBs) with an error-prone repair process known as non-homologous end joining (“NHEJ”).
  • DSBs double-stranded DNA breaks
  • NHEJ error-prone repair process
  • nucleotides may be added or removed from the DNA ends by the cell, resulting in a sequence altered from the cleaved sequence.
  • cells repair DSBs by homology-directed repair (“HDR”) or homologous recombination (“HR”) mechanisms, in which an endogenous or exogenous template can be used to direct repair of the break.
  • HDR homology-directed repair
  • HR homologous recombination
  • compositions for delivery of the protein and nucleic acid components of CRISPR/Cas to a cell, such as a cell in a patient are needed.
  • compositions for delivering mRNA encoding the CRISPR protein component, and for delivering CRISPR guide RNAs are of particular interest.
  • Compositions with useful properties for in vitro and in vivo delivery that can stabilize and deliver RNA components are also of particular interest.
  • lipid nanoparticle-based compositions with useful properties, in particular for delivery of CRISPR/Cas gene editing components.
  • the LNP compositions comprise: an RNA component; and a lipid component, wherein the lipid component comprises: (1) about 50-60 mol-% amine lipid; (2) about 8-10 mol-% neutral lipid; and (3) about 2.5-4 mol-% PEG lipid, wherein the remainder of the lipid component is helper lipid, and wherein the N/P ratio of the LNP composition is about 6,
  • the LNP compositions comprise (1) an RNA component; (2) about 50-60 mol-% amine lipid; (3) about 27-39.5 mol-% helper lipid; (4) about 8-10 mol-% neutral lipid; and (5) about 2.5-4 mol-% PEG lipid, wherein the N/P ratio of the LNP composition is about 5-7.
  • the LNP compositions comprise an RNA component and a lipid component, wherein the lipid component comprises: (1) about 50-60 mol-% amine lipid; (2) about 5-15 mol-% neutral lipid; and (3) about 2.5-4 mol-% PEG lipid, wherein the remainder of the lipid component is helper lipid, and wherein the N/P ratio of the LNP composition is about 3-10.
  • the LNP compositions comprise a lipid component that includes (1) about 40-60 mol-% amine lipid; (2) about 5-15 mol-% neutral lipid; and (3) about 2.5-4 mol-% PEG lipid, wherein the remainder of the lipid component is helper lipid, and wherein the N/P ratio of the LNP composition is about 6.
  • the LNP compositions comprise a lipid component that includes (1) about 50-60 mol-% amine, lipid; (2) about 5-15 mol-% neutral lipid; and (3) about 1.5-10 mol-% PEG lipid, wherein the remainder of the lipid component is helper lipid, and wherein the N/P ratio of the LNP composition is about 6.
  • the LNP compositions comprise an RNA component and a lipid component, wherein the lipid component comprises: (1) about 40-60 mol-% amine lipid; (2) about 0-5 mol-% neutral lipid, e,g., phospholipid; and (3) about 1.5-10 mol-% PEG lipid, wherein the remainder of the lipid component is helper lipid, and wherein the N/P ratio of the LNP composition is about 3-10.
  • the LNP compositions comprise an RNA component and a lipid component, wherein the lipid component comprises: (1) about 40-60 mol-% amine lipid; (2) less than about 1 mol-% neutral lipid, e.g., phospholipid; and (3) about 1.5-10 mol-% PEG lipid, wherein the remainder of the lipid component is helper lipid, and wherein the N/P ratio of the LNP composition is about 3-10.
  • the LNP composition is essentially free of neutral lipid.
  • the LNP compositions comprise an RNA component and a lipid component, wherein the lipid component comprises: (1) about 40-60 mol-% amine lipid; and (2) about 1.5-10 mol-% PEG lipid, wherein the remainder of the lipid component is helper lipid. wherein the N/P ratio of the LNP composition is about 3-10, and wherein the LNP composition is free of neutral lipid, e.g., phospholipid. In certain embodiments, the LNP composition is essentially free of or free of a neutral phospholipid. In certain embodiments, the LNP composition is essentially free of or free of a neutral lipid, e.g., phospholipid.
  • the RNA component comprises an mRNA, such as an RNA-guided DNA-binding agent (e.g., a Cas nuclease or Class 2 Cas nuclease).
  • the RNA component comprises a gRNA.
  • FIG. 1 shows the percentage of TTR gene editing achieved in mouse liver after delivery of CRISPR/Cas gene editing components Cas9 mRNA and gRNA in LNP compositions as indicated at a single dose of 1 mpk ( FIG. 1 A )) or 0.5 mpk ( FIG. 1 B ).
  • FIG. 2 shows particle distribution data for LNP compositions comprising Cas9 mRNA and gRNA.
  • FIG. 3 depicts physicochemical properties of LNP compositions, comparing log differential molar mass ( FIG. 3 A ) and average molecular weight measurements ( FIG. 3 B ) for the compositions.
  • FIG. 4 shows polydispersity calculations in FIG. 4 A and Burchard-Stockmeyer analysis in FIG. 4 B , analyzing the LNP compositions of FIG. 3 .
  • FIG. 5 provides the results of an experiment evaluating the effect of LNP compositions with increased PEG lipid concentrations on serum TTR knockdown, gene editing in the liver, and cytokine MCP-1 levels after a single dose administration in rats.
  • FIG. 5 A graphs serum TTR levels;
  • FIG. 5 B graphs percent editing in liver samples; and
  • FIG. 5 C provides MCP-1 levels in pg/mL.
  • FIG. 6 shows that LNP compositions maintain potency for gene editing with various PEG lipids (as measured by serum TTR levels ( FIGS. 6 A and 6 B ) and percent editing ( FIG. 6 C ).
  • FIG. 7 shows that Lipid A analogs effectively deliver gene editing cargos in LNP compositions as measured by % liver editing after a single dose administration in mouse.
  • FIG. 8 shows a dose response curve of percent editing with various LNP compositions in primary cyno hepatocytes.
  • FIG. 9 A and FIG. 9 B show serum TTR and percent editing results when the ratio of gRNA to mRNA varies
  • FIG. 9 C and FIG. 9 D show serum TTR and percent editing results in liver when the amount of Cas9 mRNA is held constant and gRNA varies following a single dose administration in mouse.
  • FIG. 10 A and FIG. 10 B show serum TTR and liver editing results after administration of LNP compositions with and without neutral lipid.
  • the present disclosure provides embodiments of lipid nanoparticle (LNP) compositions of RNAs, including CRISPR/Cas component RNAs (the “cargo”) for delivery to a cell and methods for their use.
  • the LNP compositions may exhibit improved properties as compared to prior delivery technologies.
  • the LNP composition may contain an RNA component and a lipid component, as defined herein.
  • the RNA component includes a Cas nuclease, such as a Class 2 Cas nuclease.
  • the cargo or RNA component includes an mRNA encoding a Class 2 Cas nuclease and a guide RNA or nucleic acids encoding guide RNAs.
  • the CRISPR/Cas cargo delivered via LNP formulation may include an mRNA molecule encoding a protein of interest.
  • an mRNA for expressing a protein such as green fluorescent protein (GFP), and RNA-guided DNA-binding agent, or a Cas nuclease is included.
  • LNP compositions that include a Cas nuclease snRNA, for example a Class 2 Cas nuclease mRNA that allows for expression in a cell of a Cas9 protein are provided.
  • the cargo may contain one or more guide RNAs or nucleic acids encoding guide RNAs.
  • a template nucleic acid e.g., for repair or recombination, may also be included in the composition or a template nucleic acid may be used in the methods described herein.
  • mRNA refers to a polynucleotide that comprises an open reading frame that can be translated into a polypeptide (i.e., can serve as a substrate for translation by a ribosome and amino-acylated tRNAs).
  • mRNA can comprise a phosphate-sugar backbone including ribose residues or analogs thereof, e.g., 2′-methoxy ribose residues.
  • the sugars of an mRNA phosphate-sugar backbone consist essentially of ribose residues, 2′-methoxy ribose residues, or a combination thereof.
  • mRNAs do not contain a substantial quantity of thymidine residues (e.g., 0 residues or fewer than 30, 20, 10, 5, 4, 3, or 2 thymidine residues; or less than 10%, 9%, 8%, 7%, 6%, 5%, 4%, 4%, 3%, 2%, 1%, 0.5%, 0.2%, or 0.1% thymidine content).
  • An mRNA can contain modified uridines at some or all of its uridine positions.
  • One component of the disclosed formulations is an mRNA encoding RNA-guided DNA-binding agent, such as a Cas nuclease.
  • RNA-guided DNA binding agent means a polypeptide or complex of polypeptides having RNA and DNA binding activity, or a DNA-binding subunit of such a complex, wherein the DNA binding activity is sequence-specific and depends on the sequence of the RNA.
  • RNA-guided DNA binding agents include Cas cleavases/nickases and inactivated forms thereof (“dCas DNA binding agents”).
  • dCas DNA binding agents encompasses Cas cleavases, Cas nickases, and dCas DNA binding agents.
  • Cas cleavases/nickases and dCas DNA binding agents include a Csm or Cmr complex of a type III CRISPR system, the Cas10, Csm1, or Cmr2 subunit thereof, a Cascade complex of a type 1 CRISPR system, the Cas3 subunit thereof, and Class 2 Cas nucleases.
  • a “Class 2 Cas nuclease” is a single-chain polypeptide with RNA-guided DNA binding activity.
  • Class 2 Cas nucleases include Class 2 Cas cleavases/nickases H840A, D10A, or N863A variants), which further have RNA-guided DNA cleavase or nickase activity, and Class 2 dCas DNA binding agents, in which cleavase/nickase activity is inactivated.
  • Class 2 Cas nucleases include, for example, Cas9, Cpf1, C2c1, C2c2, C2c3, HF Cas9 (e.g., N497A, R661A, Q695A, Q926A variants), HypaCas9 (e.g., N692A, M694A, Q695A, H698A variants), eSPCas9(1.0) (e.g. K810A, K1003 A, R1060A variants), and eSPCas9(1.1) (e.g., K848A, K1003A, R1060A variants) proteins and modifications thereof.
  • Cas9 Cas9
  • Cpf1, C2c1, C2c2, C2c3, HF Cas9 e.g., N497A, R661A, Q695A, Q926A variants
  • HypaCas9 e.g., N692A, M694A
  • Cpf1 protein Zetsche et al., Cell, 163: 1-13 (2015), is homologous to Cas9, and contains a RuvC-like nuclease domain.
  • Cpf1 sequences of Zetsche are incorporated by reference in their entirety. See, e.g., Zetsche, Tables S1 and S3. See, e.g., Makarova et al., Nat Rev Microbiol, 13(11): 722-36 (2015); Shmakov et al., Molecular Cell, 60:385-397 (2015).
  • the RNA-guided DNA-binding agent is a Class 2 Cas nuclease.
  • the RNA-guided DNA-binding agent has cleavase activity, which can also be referred to as double-strand endonuclease activity.
  • the RNA-guided DNA-binding agent comprises a Cas nuclease, such as a Class 2 Cas nuclease (which may be, e.g., a Cas nuclease of Type II, V, or VI).
  • Class 2 Cas nucleases include, for example, Cas9, Cpf1, C2c1, C2c2, and C2c3 proteins and modifications thereof.
  • Cas9 nucleases examples include those of the type 11 CRISPR systems of S. pyogenes, S. aureus, and other prokaryotes (see, e.g., the list in the next paragraph), and modified (e.g., engineered or mutant) versions thereof See, e.g., U.S. 2016/0312198 A1; U.S. 2016/0312199 A1.
  • Other examples of Cas nucleases include a Csm or Cmr complex of a type III CRISPR system or the Cas10, Csm1, or Cmr2 subunit thereof; and a Cascade complex of a type I CRISPR system, or the Cas3 subunit thereof.
  • the Cas nuclease may be from a Type-IIA, Type-IIB, or Type-IIC system.
  • a Type-IIA Type-IIB
  • Type-IIC Type-IIC system.
  • Makarova et al. Nat. Rev. Microbial. 9:467-477 (2011)
  • Makarova et al. Nat. Rev. Microbial, 13: 722-36 (2015)
  • Non-limiting exemplary species that the Cas nuclease can be derived from include Streptococcus pyogenes, Streptococcus thermophilus, Streptococcus sp., Staphylococcus aureus, Listeria innocua, Lactobacillus gasseri, Francisella novicida, Wolinella succinogenes, Sutterella wadsworthensis, Gammaproteobacterium, Neisseria meningitidis, Campylobacter jejuni, Pasteurella multocida, Fibrobacter succinogene, Rhodospirillum rubrum, Nocardiopsis rougevillei, Streptomyces pristinaespiralis, Streptomyces viridochromogenes, Streptomyces viridochromogenes, Streptosporangium roseum, Streptosporangium roseum, Alicyclobacillus acidocaldarius, Bacillus pseudomycoides,
  • the Cas nuclease is the Cas9 nuclease from Streptococcus pyogenes. In some embodiments, the Cas nuclease is the Cas9 nuclease from Streptococcus thermophilus. In some embodiments, the Cas nuclease is the Cas9 nuclease from Neisseria meningitidis. In some embodiments, the Cas nuclease is the Cas9 nuclease is from Staphylococcus aureus. In some embodiments, the Cas nuclease is the Cpf1 nuclease from Francisella novicida.
  • the Cas nuclease is the Cpf1 nuclease from Acidaminococcus sp. In some embodiments, the Cas nuclease is the Cpf1 nuclease from Lachnospiraceae bacterium ND2006.
  • the Cas nuclease is the Cpf1 nuclease from Francisella tularensis, Lachnospiraceae bacterium, Butyrivibrio proteoc/asticus, Peregrinibacteria bacterium, Parcubacteria bacterium, Smithella, Acidaminococcus, Ccmdidatus Alethanopiastna termitum, Eubcicterium eligens, Moraxella bovoculi, Leptospira inadai, Porphyromonas crevioricanis, Prevotella disiens, or Porphyromonas macacae.
  • the Cas nuclease is a Cpf1 nuclease from an Acidaminococcus or Lachnospiraceae.
  • Wild type Cas9 has two nuclease domains: RuvC and HNH.
  • the RuvC domain cleaves the non-target DNA strand
  • the HNH domain cleaves the target strand of DNA.
  • the Cas9 nuclease comprises more than one RuvC domain and/or more than one HNH domain.
  • the Cas9 nuclease is a wild type Cas9.
  • the Cas9 is capable of inducing a double strand break in target DNA.
  • the Cas nuclease may cleave dsDNA, it may cleave one strand of dsDNA, or it may not have DNA cleavase or nickase activity.
  • An exemplary Cas9 amino acid sequence is provided as SEQ ID NO: 3.
  • An exemplary Cas9 mRNA ORF sequence, which includes start and stop codons, is provided as SEQ ID NO: 4.
  • An exemplary Cas9 mRNA coding sequence, suitable for inclusion in a fusion protein, is provided as SEQ ID NO: 10.
  • chimeric Cas nucleases are used, where one domain or region of the protein is replaced by a portion of a different protein.
  • a Cas nuclease domain may be replaced with a domain from a different nuclease such as Fok1.
  • a Cas nuclease may be a modified nuclease.
  • the Cas nuclease may be from a Type-I CRISPR/Cas system. In some embodiments, the Cas nuclease may be a component of the Cascade complex of a Type-1 CRISPR/Cas system. In some embodiments, the Cas nuclease may be a Cas3 protein. In some embodiments, the Cas nuclease may be from a Type-III CRISPR/Cas system. In some embodiments, the Cas nuclease may have an RNA cleavage activity.
  • the RNA-guided DNA-binding agent has single-strand nickase activity, i.e., can cut one DNA strand to produce a single-strand break, also known as a “nick.”
  • the RNA-guided DNA-binding agent comprises a Cas nickase.
  • a nickase is an enzyme that creates a nick in dsDNA, i.e., cuts one strand but not the other of the DNA double helix.
  • a Cas nickase is a version of a Cas nuclease (e.g., a Cas nuclease discussed above) in which an endonucleolytic active site is inactivated, e.g., by one or more alterations (e.g., point mutations) in a catalytic domain. See, e.g., U.S. Pat. No. 8,889,356 for discussion of Cas nickases and exemplary catalytic domain alterations.
  • a Cas nickase such as a Cas9 nickase has an inactivated RuvC or HNH domain.
  • An exemplary Cas9 nickase amino acid sequence is provided as SEQ ID NO: 6.
  • An exemplary Cas9 nickase mRNA ORF sequence, which includes start and stop codons, is provided as SEQ ID NO: 7.
  • An exemplary Cas9 nickase mRNA coding sequence, suitable for inclusion in a fusion protein, is provided as SEQ ID NO: 11.
  • the RNA-guided DNA-binding agent is modified to contain only one functional nuclease domain.
  • the agent protein may be modified such that one of the nuclease domains is mutated or fully or partially deleted to reduce its nucleic acid cleavage activity.
  • a nickase is used having a RuvC domain with reduced activity.
  • a nickase is used having an inactive RuvC domain.
  • a nickase is used having an HNH domain with reduced activity.
  • a nickase is used having an inactive HNH domain.
  • a conserved amino acid within a Cas protein nuclease domain is substituted to reduce or alter nuclease activity.
  • a Cas nuclease may comprise an amino acid substitution in the RuvC or RuvC-like nuclease domain.
  • Exemplary amino acid substitutions in the RuvC or RuvC-like nuclease domain include D10A (based on the S. pyogenes Cas9 protein). See, e.g., Zetsche et al. (2015) Cell October 22:163(3): 759-771.
  • the Cas nuclease may comprise an amino acid substitution in the HNH or HNH-like nuclease domain.
  • Exemplary amino acid substitutions in the HNH or HNH-like nuclease domain include E762A, H840A, N863A, H983A, and D986A (based on the S. pyogenes Cas9 protein). See, e.g., Zetsche et al. (2015). Further exemplary amino acid substitutions include D917A, E1006A, and D1255A (based on the Francisella novicida U112 Cpf1 (FnCpf1) sequence (UniProtKB—A0Q7Q2 (CPF1_FRATN)).
  • an mRNA encoding a nickase is provided in combination with a pair of guide RNAs that are complementary to the sense and antisense strands of the target sequence, respectively.
  • the guide RNAs direct the nickase to a target sequence and introduce a DSB by generating a nick on opposite strands of the target sequence (i.e., double nicking).
  • double nicking may improve specificity and reduce off-target effects.
  • a nickase is used together with two separate guide RNAs targeting opposite strands of DNA to produce a double nick in the target DNA.
  • a nickase is used together with two separate guide RNAs that are selected to be in close proximity to produce a double nick in the target DNA.
  • the RNA-guided DNA-binding agent lacks cleavase and nickase activity.
  • the RNA-guided DNA-binding agent comprises a dCas DNA-binding polypeptide.
  • a dCas polypeptide has DNA-binding activity while essentially lacking catalytic (cleavase/nickase) activity.
  • the dCas polypeptide is a dCas9 polypeptide.
  • the RNA-guided DNA-binding agent lacking cleavase and nickase activity or the dCas DNA-binding polypeptide is a version of a Cas nuclease (e.g., a Cas nuclease discussed above) in which its endonucleolytic active sites are inactivated, e.g., by one or more alterations (e.g., point mutations) in its catalytic domains. See, e.g., U.S. 2014/0186958 A1; U.S. 2015/0166980 A1, An exemplary dCas9 amino acid sequence is provided as SEQ ID NO: 8.
  • An exemplary Cas9 mRNA ORF sequence, which includes start and stop codons, is provided as SEQ ID NO: 9.
  • An exemplary Cas9 mRNA coding sequence, suitable for inclusion in a fusion protein, is provided as SEQ ID NO: 12.
  • the RNA-guided DNA-binding agent comprises one or more heterologous functional domains (e.g., is or comprises a fusion polypeptide).
  • the heterologous functional domain may facilitate transport of the RNA-guided DNA-binding agent into the nucleus of a cell.
  • the heterologous functional domain may be a nuclear localization signal (NLS).
  • the RNA-guided DNA-binding agent may be fused with 1-10 NLS(s).
  • the RNA-guided DNA-binding agent may be fused with 1-5 NLS(s).
  • the RNA-guided DNA-binding agent may be fused with one NLS. Where one NLS is used, the NLS may be linked at the N-terminus or the C-terminus of the RNA-guided DNA-binding agent sequence.
  • the RNA-guided DNA-binding agent may be fused with more than one NLS. In some embodiments, the RNA-guided DNA-binding agent may be fused with 2, 3, 4, or 5 NLSs. In some embodiments, the RNA-guided DNA-binding agent may be fused with two NLSs. In certain circumstances, the two NLSs may be the same (e.g., two SV40 NLSs) or different. In some embodiments, the RNA-guided DNA-binding agent is fused to two SV40 NLS sequences linked at the carboxy terminus.
  • the RNA-guided DNA-binding agent may be fused with two NLSs, one linked at the N-terminus and one at the C-terminus. In some embodiments, the RNA-guided DNA-binding agent may be fused with 3 NLSs. In some embodiments, the RNA-guided DNA-binding agent may be fused with no NLS. In some embodiments, the NLS may be a monoparticle sequence, such as, e.g., the SV40 NLS, PKKKRKV or PKKKRRV. In some embodiments, the NLS may be a bipartite sequence, such as the NLS of nucleoplasmin, KRPAATKKAGQAKKKK. In a specific embodiment, a single PKKKRKV NLS may be linked at the C-terminus of the RNA-guided DNA-binding agent. One or more linkers are optionally included at the fusion site.
  • the heterologous functional domain may be capable of modifying the intracellular half-life of the RNA-guided DNA binding agent. In some embodiments, the half-life of the RNA-guided DNA binding agent may be increased. In some embodiments, the half-life of the RNA-guided DNA-binding agent may be reduced. In some embodiments, the heterologous functional domain may be capable of increasing the stability of the RNA-guided DNA-binding agent. In some embodiments, the heterologous functional domain may be capable of reducing the stability of the RNA-guided DNA-binding agent. In some embodiments, the heterologous functional domain may act as a signal peptide for protein degradation.
  • the protein degradation may be mediated by proteolytic enzymes, such as, for example, proteasomes, lysosomal proteases, or calpain proteases.
  • the heterologous functional domain may comprise a PEST sequence.
  • the RNA-guided DNA-binding agent may be modified by addition of ubiquitin or a polyubiquitin chain.
  • the ubiquitin may be a ubiquitin-like protein (UBL).
  • Non-limiting examples of ubiquitin-like proteins include small ubiquitin-like modifier (SUMO), ubiquitin cross-reactive protein (UCRP, also known as interferon-stimulated gene-15 (ISG15)), ubiquitin-related modifier-1 (URM1), neuronal-precursor-cell-expressed developmentally downregulated protein-8 (NEDD8, also called Rub1 in S. cerevislee ), human leukocyte antigen F-associated (FAT10), autophagy-8 (ATG8) and -12 (ATG12), Fau ubiquitin-like protein (FUB1), membrane-anchored UBL (MUB), ubiquitin fold-modifier-1 (UFM1), and ubiquitin-like protein-5 (UBL5).
  • SUMO small ubiquitin-like modifier
  • URP ubiquitin cross-reactive protein
  • ISG15 interferon-stimulated gene-15
  • UDM1 ubiquitin-related modifier-1
  • NEDD8 neuronal-precursor-cell-
  • the heterologous functional domain may be a marker domain.
  • marker domains include fluorescent proteins, purification tags, epitope tags, and reporter gene sequences.
  • the marker domain may he a fluorescent protein.
  • suitable fluorescent proteins include green fluorescent proteins (e.g., GFP, GFP-2, tagGFP, turboGFP, sfGFP, EGFP, Emerald, Azami Green, Monomeric Azami Green, CopGFP, AceGFP, ZsGreen1), yellow fluorescent proteins (e.g., YFP, EYFP, Citrine, Venus, YPet, PhiYFP, ZsYellow1), blue fluorescent proteins (e.g., EBFP, EBFP2, Azurite, mKalamal, GFPuv, Sapphire, T-sapphire,), cyan fluorescent proteins (e.g., ECFP, Cerulean, CyPet, AmCyan1, Midoriishi-Cyan), red fluorescent proteins (e.
  • the marker domain may be a purification tag and/or an epitope tag.
  • Non-limiting exemplary tags include glutathione-S-transferase (GST), chitin binding protein (CBP), maltose binding protein (MBP), thioredoxin (TRX), poly(NANP), tandem affinity purification (TAP) tag, myc, AcV5, AU1, AU5, E, ECS, E2, FLAG, HA, nus, Softag 1, Softag 3, Strep, SBP, Glu-Glu, HSV, KT3, S, S1, T7, V5, VSV-G, 6 ⁇ His, 8 ⁇ His, biotin carboxyl carrier protein (BCCP), poly-His, and calmodulin.
  • GST glutathione-S-transferase
  • CBP chitin binding protein
  • MBP maltose binding protein
  • TRX thioredoxin
  • poly(NANP) tandem affinity purification
  • TAP tandem affinity pur
  • Non-limiting exemplary reporter genes include glutathione-S-transferase (GST), horseradish peroxidase (HRP), chloramphenicol acetyltransferase (CAT), beta-galactosidase, beta-glucuronidase, luciferase, or fluorescent proteins.
  • GST glutathione-S-transferase
  • HRP horseradish peroxidase
  • CAT chloramphenicol acetyltransferase
  • beta-galactosidase beta-glucuronidase
  • luciferase or fluorescent proteins.
  • the heterologous functional domain may target the RNA-guided DNA-binding agent to a specific organelle, cell type, tissue, or organ. In some embodiments, the heterologous functional domain may target the RNA-guided DNA-binding agent to mitochondria.
  • the heterologous functional domain may be an effector domain.
  • the effector domain may modify or affect the target sequence.
  • the effector domain may be chosen from a nucleic acid binding domain, a nuclease domain (e.g., a non-Cas nuclease domain), an epigenetic modification domain, a transcriptional activation domain, or a transcriptional repressor domain.
  • the heterologous functional domain is a nuclease, such as a FokI nuclease.
  • the heterologous functional domain is a transcriptional activator or repressor.
  • a transcriptional activator or repressor See, e.g., Qi et al., “Repurposing CRISPR as an RNA-guided platform for sequence-specific control of gene expression,” Cell 152:1173-83 (2013); Perez-Pinera et al., “RNA-guided gene activation by CRISPR-Cas9-based transcription factors,” Nat. Methods 10:973-6 (2013); Mali et al., “CAS9 transcriptional activators for target specificity screening and paired nickases for cooperative genome engineering,” Nat. Biotechnol.
  • the RNA-guided DNA-binding agent essentially becomes a transcription factor that can be directed to bind a desired target sequence using a guide RNA.
  • the DNA modification domain is a methylation domain, such as a demethylation or methyltransferase domain.
  • the effector domain is a DNA modification domain, such as a base-editing domain.
  • the DNA modification domain is a nucleic acid editing domain that introduces a specific modification into the DNA, such as a deaminase domain, See, e.g., WO 2015/089406; U.S. 2016/0304846.
  • the nucleic acid editing domains, deaminase domains, and Cas9 variants described in WO 2015/089406 and U.S. 2016/0304846 are hereby incorporated by reference.
  • the nuclease may comprise at least one domain that interacts with a guide RNA (“gRNA”). Additionally, the nuclease may be directed to a target sequence by a gRNA. In Class 2 Cas nuclease systems, the gRNA interacts with the nuclease as well as the target sequence, such that it directs binding to the target sequence. In some embodiments, the gRNA provides the specificity for the targeted cleavage, and the nuclease may be universal and paired with different gRNAs to cleave different target sequences. Class 2 Cas nuclease may pair with a gRNA scaffold structure of the types, orthologs, and exemplary species listed above.
  • gRNA Guide RNA
  • the cargo for the LNP formulation includes at least one gRNA.
  • the gRNA may guide the Cas nuclease or Class 2 Cas nuclease to a target sequence on a target nucleic acid molecule.
  • a gRNA binds with and provides specificity of cleavage by a Class 2 Cas nuclease.
  • the gRNA and the Cas nuclease may form a ribonucleoprotein (RNP), e.g., a CRISPR/Cas complex such as a CRISPR/Cas9 complex which may be delivered by the LNP composition.
  • RNP ribonucleoprotein
  • the CRISPR/Cas complex may be a Type-II CRISPR/Cas9 complex. In some embodiments, the CRISPR/Cas complex may be a Type-V CRISPR/Cas complex, such as a Cpf1/guide RNA complex. Cas nucleases and cognate gRNAs may be paired. The gRNA scaffold structures that pair with each Class 2 Cas nuclease vary with the specific CRISPR/Cas system.
  • RNA Ribonucleic acid
  • gRNA gRNA
  • tracrRNA RNA trRNA
  • the crRNA and trRNA may be associated as a single RNA molecule (single guide RNA, sgRNA) or in two separate RNA molecules (dual guide RNA, dgRNA).
  • sgRNA single guide RNA
  • dgRNA dual guide RNA
  • gRNA dual guide RNA
  • the trRNA may be a naturally-occurring sequence, or a trRNA sequence with modifications or variations compared to naturally-occurring sequences.
  • a “guide sequence” refers to a sequence within a guide RNA that is complementary to a target sequence and functions to direct a guide RNA to a target sequence for binding or modification (e.g., cleavage) by an RNA-guided DNA binding agent.
  • a “guide sequence” may also be referred to as a “targeting sequence,” or a “spacer sequence.”
  • a guide sequence can be 20 base pairs in length, e.g., in the case of Streptococcus pyogenes (i.e., Spy Cas9) and related Cas9 homologs/orthologs.
  • the target sequence is in a gene or on a chromosome, for example, and is complementary to the guide sequence.
  • the degree of complementarity or identity between a guide sequence and its corresponding target sequence may be about 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%.
  • the guide sequence and the target region may be 100% complementary or identical. In other embodiments, the guide sequence and the target region may contain at least one mismatch.
  • the guide sequence and the target sequence may contain 1, 2, 3, or 4 mismatches, where the total length of the target sequence is at least 17, 18, 19, 20 or more base pairs.
  • the guide sequence and the target region may contain 1-4 mismatches where the guide sequence comprises at least 17, 18, 19, 20 or more nucleotides.
  • the guide sequence and the target region may contain 1, 2, 3, or 4 mismatches where the guide sequence comprises 20 nucleotides.
  • Target sequences for Cas proteins include both the positive and negative strands of genomic DNA (i.e., the sequence given and the sequence's reverse compliment), as a nucleic acid substrate for a Cas protein is a double stranded nucleic acid. Accordingly, where a guide sequence is said to be “complementary to a target sequence”, it is to be understood that the guide sequence may direct a guide RNA to bind to the reverse complement of a target sequence. Thus, in some embodiments, where the guide sequence binds the reverse complement of a target sequence, the guide sequence is identical to certain nucleotides of the tareet sequence (e.g., the target sequence not including the PAM) except for the substitution of U for T in the guide sequence.
  • the length of the targeting sequence may depend on the CRISPR/Cas system and components used. For example, different Class 2 Cas nucleases from different bacterial species have varying optimal targeting sequence lengths. Accordingly, the targeting sequence may comprise 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, or more than 50 nucleotides in length. In some embodiments, the targeting sequence length is 0, 1, 2, 3, 4, or 5 nucleotides longer or shorter than the guide sequence of a naturally-occurring CRISPR/Cas system. In certain embodiments, the Cas nuclease and gRNA scaffold will be derived from the same CRISPR/Cas system. In some embodiments, the targeting sequence may comprise or consist of 18-24 nucleotides. In some embodiments, the targeting sequence may comprise or consist of 19-21 nucleotides. In some embodiments, the targeting sequence may comprise or consist of 20 nucleotides.
  • Certain embodiments of the invention also provide nucleic acids, e.g., expression cassettes, encoding the gRNA described herein.
  • a “guide RNA nucleic acid” is used herein to refer to a guide RNA (e.g. an sgRNA or a dgRNA) and a guide RNA expression cassette, which is a nucleic acid that encodes one or more guide RNAs.
  • the nucleic acid may be a DNA molecule. In some embodiments, the nucleic acid may comprise a nucleotide sequence encoding a crRNA. In some embodiments, the nucleotide sequence encoding the crRNA comprises a targeting sequence flanked by all or a portion of a repeat sequence from a naturally-occurring CRISPR/Cas system. In some embodiments, the nucleic acid may comprise a nucleotide sequence encoding a tracr RNA. In some embodiments, the crRNA and the tracr RNA may be encoded by two separate nucleic acids. In other embodiments, the crRNA and the tracr RNA may be encoded by a single nucleic acid.
  • the crRNA and the tracr RNA may be encoded by opposite strands of a single nucleic acid. In other embodiments, the crRNA and the tracr RNA may be encoded by the same strand of a single nucleic acid.
  • the gRNA nucleic acid encodes an sgRNA. In some embodiments, the gRNA nucleic acid encodes a Cas9 nuclease sgRNA. In some embodiments, the gRNA nucleic acid encodes a Cpf1 nuclease sgRNA.
  • the nucleotide sequence encoding the guide RNA may be operably linked to at least one transcriptional or regulatory control sequence, such as a promoter, a 3′ UTR, or a 5′ UTR.
  • the promoter may be a tRNA promoter, e.g, tRNA Lys3 , or a tRNA chimera, See Mefferd et al., RNA, 2015 21:1683-9; Scherer et al., Nucleic Acids Res. 2007 35: 2620-2628.
  • the promoter may be recognized by RNA polymerase III (Pol III).
  • Non-limiting examples of Pol III promoters also include U6 and H1 promoters.
  • the nucleotide sequence encoding the guide RNA may be operably linked to a mouse or human U6 promoter.
  • the gRNA nucleic acid is a modified nucleic acid.
  • the gRNA nucleic acid includes a modified nucleoside or nucleotide.
  • the gRNA nucleic acid includes a 5′ end modification, for example a modified nucleoside or nucleotide to stabilize and prevent integration of the nucleic acid.
  • the gRNA nucleic acid comprises a double-stranded DNA having a 5′ end modification on each strand.
  • the gRNA nucleic acid includes an inverted dideoxy-T or an inverted abasic nucleoside or nucleotide as the 5′ end modification.
  • the gRNA nucleic acid includes a label such as biotin, desthiobioten-TEG, digoxigenin, and fluorescent markers, including, for example, FAM, ROX, TAMRA, and AlexaFluor.
  • more than one gRNA nucleic acid such as a gRNA
  • a CRISPR/Cas nuclease system can be used with a CRISPR/Cas nuclease system.
  • Each gRNA nucleic acid may contain a different targeting sequence, such that the CRISPR/Cas system cleaves more than one target sequence.
  • one or more gRNAs may have the same or differing properties such as activity or stability within a CRISPR/Cas complex. Where more than one gRNA is used, each gRNA can be encoded on the same or on different gRNA nucleic acid.
  • the promoters used to drive expression of the more than one gRNA may be the same or different.
  • the LNP compositions comprise modified RNAs.
  • Modified nucleosides or nucleotides can be present in an RNA, for example a gRNA or mRNA.
  • a gRNA or mRNA comprising one or more modified nucleosides or nucleotides, for example, is called a “modified” RNA to describe the presence of one or more non-naturally and/or naturally occurring components or configurations that are used instead of or in addition to the canonical A, G, C, and U residues.
  • a modified RNA is synthesized with a non-canonical nucleoside or nucleotide, here called “modified.”
  • Modified nucleosides and nucleotides can include one or more of: (i) alteration, e.g., replacement, of one or both of the non-linking phosphate oxygens and/or of one or more of the linking phosphate oxygens in the phosphodiester backbone linkage (an exemplary backbone modification); (ii) alteration, e.g., replacement, of a constituent of the ribose sugar, e.g., of the 2′ hydroxyl on the ribose sugar (an exemplary sugar modification); (iii) wholesale replacement of the phosphate moiety with “dephospho” linkers (an exemplary backbone modification); (iv) modification or replacement of a naturally occurring nucleobase, including with a non-canonical nucleobase (an exemplary base modification); (v) replacement or modification of the ribose-phosphate backbone (an exemplary backbone modification); (vi) modification of the 3′ end or 5′ end of the oligonucleotide,
  • Certain embodiments comprise a 5′ end modification to an mRNA, gRNA, or nucleic acid. Certain embodiments comprise a 3′ end modification to an mRNA, gRNA, or nucleic acid. A modified RNA can contain 5′ end and 3′ end modifications. A modified RNA can contain one or more modified residues at non-terminal locations. In certain embodiments, a gRNA includes at least one modified residue. In certain embodiments, an mRNA includes at least one modified residue.
  • a first sequence is considered to “comprise a sequence with at least X % identity to” a second sequence if an alignment of the first sequence to the second sequence shows that X % or more of the positions of the second sequence in its entirety are matched by the first sequence.
  • the sequence AAGA comprises a sequence with 100% identity to the sequence AAG because an alignment would give 100% identity in that there are matches to all three positions of the second sequence.
  • sequence 5′-AXG where X is any modified uridine, such as pseudouridine, N1-methyl pseudouridine, or 5-methoxyuridine, is considered 100% identical to AUG in that both are perfectly complementary to the same sequence (5′-CAU).
  • exemplary alignment algorithms are the Smith-Waterman and Needleman-Wunsch algorithms, which are well-known in the art.
  • Needleman-Wunsch algorithm with default settings of the Needleman-Wunsch algorithm interface provided by the EBI at the www.ebi.ac.uk web server is generally appropriate.
  • composition or formulation disclosed herein comprises an mRNA comprising an open reading frame (ORF) encoding an RNA-guided DNA binding agent, such as a Cas nuclease, or Class 2 Cas nuclease as described herein.
  • ORF open reading frame
  • an mRNA comprising an ORF encoding an RNA-guided DNA binding agent, such as a Cas nuclease or Class 2 Cas nuclease is provided, used, or administered.
  • the ORF encoding an RNA-guided DNA binding agent is a “modified RNA-guided DNA binding agent ORF” or simply a “modified ORF,” which is used as shorthand to indicate that the ORF is modified in one or more of the following ways: (1) the modified ORF has a uridine content ranging from its minimum uridine content to 150% of the minimum uridine content; (2) the modified ORF has a uridine dinucleotide content ranging from its minimum uridine dinucleotide content to 150% of the minimum uridine dinucleotide content; (3) the modified ORF has at least 90% identity to any one of SEQ ID NOs: 1, 4, 7, 9, 10, 11, 12, 14, 15, 17, 18, 20, 21, 23, 24, 26, 27, 29, 30, 50, 52, 54, 65, or 66; (4) the modified ORF consists of a set of codons of which at least 75% of the codons are minimal uridine codon(s) for a given amino acid, e.g.
  • the modified ORF comprises at least one modified uridine.
  • the modified ORF is modified in at least two, three, or four of the foregoing ways.
  • the modified ORF comprises at least one modified uridine and is modified in at least one, two, three, or all of (1)-(4) above.
  • Modified uridine is used herein to refer to a nucleoside other than thymidine with the same hydrogen bond acceptors as uridine and one or more structural differences from uridine.
  • a modified uridine is a substituted uridine, i.e., a uridine in which one or more non-proton substituents (e.g., alkoxy, such as methoxy) takes the place of a proton.
  • a modified uridine is pseudouridine.
  • a modified uridine is a substituted pseudouridine, i.e., a pseudouridine in which one or more non-proton substituents (e.g., alkyl, such as methyl) takes the place of a proton.
  • a modified uridine is any of a substituted uridine, pseudouridine, or a substituted pseudouridine.
  • the modified ORF may consist of a set of codons of which at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% of the codons are codons listed in the Table of Minimal Uridine Codons.
  • the modified ORF may comprise a sequence with at least 90%, 95%, 98%, 99%, or 100% identity to any one of SEQ ID NO: 1, 4, 7, 9, 10, 11, 12, 14, 15, 17, 18, 20, 21, 23, 24, 26, 27, 29, 30, 50, 52, 54, 65, or 66.
  • the modified ORF may comprise a sequence with at least 90%, 95%, 98%, 99%, or 100% identity to any one of SEQ ID NO: 1, 4, 7, 9, 10, 11, 12, 14, 15, 17, 18, 20, 21, 23, 24, 26, 27, 29, 30, 50, 52, 54, 65, or 66.
  • the modified ORF may have a uridine content ranging from its minimum uridine content to 150%, 145%, 140%, 135%, 130%, 125%, 120%,115%, 110%, 105%, 104%, 103%, 102%, or 101% of the minimum uridine content.
  • the modified ORF may have a uridine dinucleotide content ranging from its minimum uridine dinucleotide content to 150%, 145%, 140%, 135%, 130%, 125%, 120%, 115%, 110%, 105%, 104%, 103%, 102%, or 101% of the minimum uridine dinucleotide content.
  • the modified ORF may comprise a modified uridine at least at one, a plurality of, or all uridine positions.
  • the modified uridine is a uridine modified at the 5 position, e.g., with a halogen, methyl, or ethyl.
  • the modified uridine is a pseudouridine modified at the I position, e.g., with a halogen, methyl, or ethyl.
  • the modified uridine can be, for example, pseudouridine, N1-methyl-pseudouridine, 5-methoxyuridine, 5-iodouridine, or a combination thereof.
  • the modified uridine is 5-methoxyuridine. In some embodiments, the modified uridine is 5-iodouridine. In some embodiments, the modified uridine is pseudouridine. In some embodiments, the modified uridine is N1-methyl-pseudouridine. In some embodiments, the modified uridine is a combination of pseudouridine and N1-methyl-pseudouridine. In some embodiments, the modified uridine is a combination of pseudouridine and 5-methoxyuridine. In some embodiments, the modified uridine is a combination of N1-methyl pseudouridine and 5-methoxyuridine.
  • the modified uridine is a combination of 5-iodouridine and N1-methyl-pseudouridine. In some embodiments, the modified uridine is a combination of pseudouridine and 5-iodouridine. In some embodiments, the modified uridine is a combination of 5-iodouridine and 5-methoxyuridine.
  • At least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% of the uridine positions in an mRNA according to the disclosure are modified uridines.
  • 10%-25%, 15-25%, 25-35%, 35-45%, 45-55%, 55-65%, 65-75%, 75-85%, 85-95%, or 90-100% of the uridine positions in an mRNA according to the disclosure are modified uridines, e.g., 5-methoxyuridine, 5-iodouridine, N1-methyl pseudouridine, pseudouridine, or a combination thereof.
  • 10%-25%, 15-25%, 25-35%, 35-45%, 45-55%, 55-65%, 65-75%, 75-85%, 85-95%, or 90-100% of the uridine positions in an mRNA, according to the disclosure are 5-methoxyuridine. In some embodiments, 10%-25%, 15-25%, 25-35%, 35-45%, 45-55%, 55-65%, 65-75%, 75-85%, 85-95%, or 90-100% of the uridine positions in an mRNA according to the disclosure are pseudouridine.
  • 10%-25%, 15-25%, 25-35%, 35-45%, 45-55%, 55-65%, 65-75%, 75-85%, 85-95%, or 90-100% of the uridine positions in an mRNA according to the disclosure are N1-methyl pseudouridine. In some embodiments, 10%-25%, 15-25%, 25-35%, 35-45%, 45-55%, 55-65%, 65-75%, 75-85%, 85-95%, or 90-100% of the uridine positions in an mRNA according to the disclosure are 5-iodouridine.
  • 10%-25%, 15-25%, 25-35%, 35-45%, 45-55%, 55-65%, 65-75%, 75-85%, 85-95%, or 90-100% of the uridine positions in an mRNA according to the disclosure are 5-methoxyuridine, and the remainder are N1-methyl pseudouridine.
  • 10%-25%, 15-25%, 25-35%, 35-45%, 45-55%, 55-65%, 65-75%, 75-85%, 85-95%, or 90-100% of the uridine positions in an mRNA according to the disclosure are 5-iodouridine, and the remainder are N1-methyl pseudouridine.
  • the modified ORF may comprise a reduced uridine dinucleotide content, such as the lowest possible uridine dinucleotide (UU) content, e.g. an ORF that (a) uses a minimal uridine codon (as discussed above) at every position and (b) encodes the same amino acid sequence as the given ORF.
  • UU uridine dinucleotide
  • the uridine dinucleotide (UU) content can be expressed in absolute terms as the enumeration of UU dinucleotides in an ORF or on a rate basis as the percentage of positions occupied by the uridines of uridine dinucleotides (for example, AUUAU would have a uridine dinucleotide content of 40% because 2 of 5 positions are occupied by the uridines of a uridine dinucleotide).
  • Modified uridine residues are considered equivalent to uridines for the purpose of evaluating minimum uridine dinucleotide content.
  • the mRNA comprises at least one UTR from an expressed mammalian mRNA, such as a constitutively expressed mRNA.
  • An mRNA is considered constitutively expressed in a mammal if it is continually transcribed in at least one tissue of a healthy adult mammal.
  • the mRNA comprises a 5′ UTR, 3′ UTR, or 5′ and 3′ UTRs from an expressed mammalian RNA, such as a constitutively expressed mammalian mRNA. Actin mRNA is an example of a constitutively expressed mRNA.
  • the mRNA comprises at least one UTR from Hydroxysteroid 17-Beta Dehydrogenase 4 (HSD17B4 or HSD), e.g., a 5′ UTR from HSD.
  • the mRNA comprises at least one UTR from a globin mRNA, for example, human alpha globin (HBA) mRNA, human beta globin (HBB) mRNA, or Xenopus laevis beta globin (XBG) mRNA.
  • the mRNA comprises a 5′ UTR, 3′ UTR, or 5′ and 3′ UTRs from a globin mRNA, such as HBA, HBB, or XBG.
  • the mRNA comprises a 5′ UTR from bovine growth hormone, cytomegalovirus (CMV), mouse Hba-a1, HSD, an albumin gene, HBA, HBB, or XBG. In some embodiments, the mRNA comprises a 3′ UTR from bovine growth hormone, cytomegalovirus, mouse Hba-a1, HSD, an albumin gene, HBA, HBB, or XBG.
  • the mRNA comprises 5′ and 3′ UTRs from bovine growth hormone, cytomegalovirus, mouse Hba-a1, HSD, an albumin gene, HBA, HBB, XBG, heat shock protein 90 (Hsp90), glyceraldehyde 3-phosphate dehydrogenase (GAPDH), beta-actin, alpha-tubulin, tumor protein (p53), or epidermal growth factor receptor (EGFR).
  • bovine growth hormone cytomegalovirus
  • mouse Hba-a1, HSD an albumin gene
  • HBA HBB
  • XBG heat shock protein 90
  • Hsp90 heat shock protein 90
  • GPDH glyceraldehyde 3-phosphate dehydrogenase
  • beta-actin beta-actin
  • alpha-tubulin alpha-tubulin
  • tumor protein p53
  • EGFR epidermal growth factor receptor
  • the mRNA comprises 5′ and 3′ UTRs that are from the same source, e.g., a constitutively expressed mRNA such as actin, albumin, or a globin such as HBA, HBB, or XBG.
  • a constitutively expressed mRNA such as actin, albumin, or a globin such as HBA, HBB, or XBG.
  • the mRNA does not comprise a 5′ UTR, e.g., there are no additional nucleotides between the 5′ cap and the start codon.
  • the mRNA comprises a Kozak sequence (described below) between the 5′ cap and the start codon, but does not have any additional 5′ UTR.
  • the mRNA does not comprise a 3′ UTR, e.g., there are no additional nucleotides between the stop codon and the poly-A tail.
  • the mRNA comprises a Kozak sequence.
  • the Kozak sequence can affect translation initiation and the overall yield of a polypeptide translated from an mRNA.
  • a Kozak sequence includes a methionine codon that can function as the start codon.
  • a minimal Kozak sequence is NNNRUGN wherein at least one of the following is true: the first N is A or G and the second N is G.
  • R means a purine (A or G).
  • the Kozak sequence is RNNRUGN, NNNRUGG, RNNRUGG, RNNAUGN, NNNAUGG, or RNNAUGG.
  • the Kozak sequence is rccRUGg with zero mismatches or with up to one or two mismatches to positions in lowercase. In some embodiments, the Kozak sequence is rccAUGg with zero mismatches or with up to one or two mismatches to positions in lowercase. In some embodiments, the Kozak sequence is gccRccAUGG with zero mismatches or with up to one, two, or three mismatches to positions in lowercase. In some embodiments, the Kozak sequence is gccAccAUG with zero mismatches or with up to one, two, three, or four mismatches to positions in lowercase. In some embodiments, the Kozak sequence is GCCACCAUG. In some embodiments, the Kozak sequence is gccgccRccAUGG with zero mismatches or with up to one, two, three, or four mismatches to positions in lowercase.
  • the mRNA comprising an ORF encoding an RNA-guided DNA binding agent comprises a sequence having at least 90% identity to SEQ ID NO: 43, optionally wherein the ORF of SEQ ID NO: 43 (i.e., SEQ ID NO: 4) is substituted with an alternative ORF of any one of SEQ ID NO: 7, 9, 10, 11, 12, 14, 15, 17, 18, 20, 21, 23, 24, 26, 27, 29, 30, 50, 52, 54, 65, or 66.
  • the mRNA comprising an ORF encoding an RNA-guided DNA binding agent comprises a sequence having at least 90% identity to SEQ ID NO: 44, optionally wherein the ORF of SEQ ID NO: 44 (i.e., SEQ ID NO: 4) is substituted with an alternative ORF of any one of SEQ ID NO: 7, 9, 10, 11, 12, 14, 15, 17, 18, 20, 21, 23, 24, 26, 27, 29, 30, 50, 52, 54, 65, or 66.
  • the mRNA comprising an ORF encoding an RNA-guided DNA binding agent comprises a sequence having at least 90% identity to SEQ ID NO: 56, optionally wherein the ORF of SEQ ID NO: 56 (i.e., SEQ ID NO: 4) is substituted with an alternative ORF of any one of SEQ ID NO: 7, 9, 10, 11, 12, 14, 15, 17, 18, 20, 21, 23, 24, 26, 27, 29, 30, 50, 52, 54, 65, or 66.
  • the mRNA comprising an ORF encoding an RNA-guided DNA binding agent comprises a sequence having at least 90% identity to SEQ ID NO: 57, optionally wherein the ORF of SEQ ID NO: 57 (i.e., SEQ ID NO: 4) is substituted with an alternative ORF of any one of SEQ ID NO: 7, 9, 10, 11, 12, 14, 15, 17, 18, 20, 21, 23, 24, 26, 27, 29, 30, 50, 52, 54, 65, or 66.
  • the mRNA comprising an ORF encoding an RNA-guided DNA binding agent comprises a sequence having at least 90% identity to SEQ ID NO: , optionally wherein the ORF of SEQ ID NO: 58 (i.e., SEQ ID NO: 4) is substituted with an alternative ORF of any one of SEQ ID NO: 7, 9, 10, 11, 12, 14, 15, 17, 18, 20, 21, 23, 24, 26, 27, 29, 30, 50, 52, 54, 65, or 66.
  • the mRNA comprising an ORF encoding an RNA-guided DNA binding agent comprises a sequence having at least 90% identity to SEQ ID NO: 59, optionally wherein the ORE of SEQ ID NO: 59 (i.e., SEQ ID NO: 4) is substituted with art alternative ORF of any one of SEQ ID NO: 7, 9, 10, 11, 12, 14, 15, 17, 18, 20, 21, 23, 24, 26, 27, 29, 30, 50, 52, 54, 65, or 66.
  • the mRNA comprising an ORF encoding an RNA-guided DNA binding agent comprises a sequence having at least 90% identity to SEQ ID NO: 60, optionally wherein the ORF of SEQ ID NO: 60 (i.e., SEQ ID NO: 4) is substituted with an alternative ORE of any one of SEQ ID NO: 7, 9, 10, 11, 12, 14, 15, 17, 18, 20, 21, 23, 24, 26, 27, 29, 30, 50, 52, 54, 65, or 66.
  • the mRNA comprising an ORF encoding an RNA-guided DNA binding agent comprises a sequence having at least 90% identity to SEQ ID NO: 61, optionally wherein the ORF of SEQ ID NO: 61 (i.e., SEQ ID NO: 4) is substituted with an alternative ORF of any one of SEQ ID NO: 7, 9, 10, 11, 12, 14, 15, 17, 18, 20, 21, 23, 24, 26, 27, 29, 30, 50, 52, 54, 65, or 66.
  • the mRNA comprises an alternative ORF of any one of SEQ ID NO: 7, 9, 10, 11, 12, 14, 15, 17, 18, 20, 21, 23, 24, 26, 27, 29, 30, 50, 52, 54, 65, or 66.
  • the degree of identity to the optionally substituted sequences of SEQ ID NOs 43, 44, or 56-61 is 95%. In some embodiments, the degree of identity to the optionally substituted sequences of SEQ ID NOs 43, 44, or 56-61 is 98%. In some embodiments, the degree of identity to the optionally substituted sequences of SEQ ID NOs 43, 44, or 56-61 is 99%. In some embodiments, the degree of identity to the optionally substituted sequences of SEQ ID NOs 43, 44, or 56-61 is 100%.
  • an mRNA disclosed herein comprises a 5′ cap, such as a Cap0, Cap1, or Cap2.
  • a 5′ cap is generally a 7-methylguanine ribonucleotide (which may be further modified, as discussed below e.g. with respect to ARCA) linked through a 5′-triphosphate to the 5′ position of the first nucleotide of the 5′-to-3′ chain of the mRNA, i.e., the first cap-proximal nucleotide.
  • the riboses of the first and second cap-proximal nucleotides of the mRNA both comprise a 2′-hydroxyl.
  • the riboses of the first and second transcribed nucleotides of the mRNA comprise a 2′-methoxy and a 2′-hydroxyl, respectively.
  • the riboses of the first and second cap-proximal nucleotides of the mRNA both comprise a 2′-methoxy. See, e.g., Katibah et al. (2014) Proc Natl Acad Sci USA 111(33):12025-30; Abbas et al. (2017) Proc Natl Acad Sci USA 114(11):E2106-E2115.
  • Most endogenous higher eukaryotic mRNAs, including mammalian mRNAs such as human mRNAs, comprise Cap1 or Cap2.
  • Cap0 and other cap structures differing from Cap1 and Cap2 may be immunogenic in mammals, such as humans, due to recognition as “non-self” by components of the innate immune system such as IFIT-1 and IFIT-5, which can result in elevated cytokine levels including type I interferon.
  • components of the innate immune system such as IFIT-1 and IFIT-5 may also compete with eIF4E for binding of an mRNA with a cap other than Cap1 or Cap2, potentially inhibiting translation of the mRNA.
  • a cap can be included co-transcriptionally.
  • ARCA anti-reverse cap analog; Thermo Fisher Scientific Cat. No. AM8045
  • ARCA is a cap analog comprising a 7-methylguanine 3′-methoxy-5′-triphosphate linked to the 5′ position of a guanine ribonucleotide which can be incorporated in vitro into a transcript at initiation.
  • ARCA results in a Cap0 cap in which the 2′ position of the first cap-proximal nucleotide is hydroxyl.
  • CleanCapTM AG (m7G(5′)ppp(5′)(2′OMeA)pG; TriLink Biotechnologies Cat. No. N-7113) or CleanCapTM GG (m7G(5′)ppp(5′)(2′OMeG)pG; TriLink Biotechnologies Cat. No. N-7133) can be used to provide a Cap1 structure co-transcriptionally, 3′-O-methylated versions of ClearCapTM AG and CleanCapTM GG are also available from TriLink Biotechnologies as Cat. Nos. N-7413 and N-7433, respectively.
  • the CleanCapTM AG structure is shown below.
  • a cap can be added to an RNA post-transcriptionally.
  • Vaccinia capping enzyme is commercially available (New England Biolabs Cat. No. M2080S) and has RNA triphosphatase and guanylyttransferase activities, provided by its D1 subunit, and guanine methyltransferase, provided by its D12 subunit.
  • it can add a 7-methylguanine to an RNA, so as to give Cap0, in the presence of S-adenosyl methionine and GTP. See, e.g., Guo, P, and Moss, B. (1990) Proc. Natl. Acad. Sci. USA 87, 4023-4027; Mao, X. and Shuman, S. (1994) J. Biol. Chem. 269, 24172-24479.
  • the mRNA further comprises a poly-adenylated (poly-A) tail.
  • the poly-A tail comprises at least 20, 30, 40, 50, 60, 70, 80, 90, or 100 adenines, optionally up to 300 adenines.
  • the poly-A tail comprises 95, 96, 97, 98, 99, or 100 adenine nucleotides.
  • the poly-A tail is “interrupted” with one or more non-adenine nucleotide “anchors” at one or more locations within the poly-A tail.
  • the poly-A tails may comprise at least 8 consecutive adenine nucleotides, but also comprise one or more non-adenine nucleotide.
  • “non-adenine nucleotides” refer to any natural or non-natural nucleotides that do not comprise adenine. Guanine, thymine, and cytosine nucleotides are exemplary non-adenine nucleotides.
  • the poly-A tails on the mRNA described herein may comprise consecutive adenine nucleotides located 3′ to nucleotides encoding an RNA-guided DNA-binding agent or a sequence of interest.
  • the poly-A tails on mRNA comprise non-consecutive adenine nucleotides located 3′ to nucleotides encoding an RNA-guided DNA-binding agent or a sequence of interest, wherein non-adenine nucleotides interrupt the adenine nucleotides at regular or irregularly spaced intervals.
  • the mRNA further comprises a poly-adenylated (poly-A) tail.
  • the poly-A tail comprises at least 20, 30, 40, 50, 60, 70, 80, 90, or 100 adenines, optionally up to 300 adenines, In some embodiments, the poly-A tail comprises 95, 96, 97, 98, 99, or 100 adenine nucleotides. In some instances, the poly-A tail is “interrupted” with one or more non-adenine nucleotide “anchors” at one or more locations within the poly-A tail.
  • the poly-A tails may comprise at least 8 consecutive adenine nucleotides, but also comprise one or more non-adenine nucleotide.
  • “non-adenine nucleotides” refer to any natural or non-natural nucleotides that do not comprise adenine. Guanine, thymine, and cytosine nucleotides are exemplary non-adenine nucleotides.
  • the poly-A tails on the mRNA described herein may comprise consecutive adenine nucleotides located 3′ to nucleotides encoding an RNA-guided DNA-binding agent or a sequence of interest.
  • the poly-A tails on mRNA comprise non-consecutive adenine nucleotides located 3′ to nucleotides encoding an RNA-guided DNA-binding agent or a sequence of interest, wherein non-adenine nucleotides interrupt the adenine nucleotides at regular or irregularly spaced intervals.
  • the one or more non-adenine nucleotides are positioned to interrupt the consecutive adenine nucleotides so that a poly(A) binding protein can bind to a stretch of consecutive adenine nucleotides.
  • one or more non-adenine nucleotide(s) is located after at least 8, 9, 10, 11, or 12 consecutive adenine nucleotides.
  • the one or more non-adenine nucleotide is located after at least 8-50 consecutive adenine nucleotides.
  • the one or more non-adenine nucleotide is located after at least 8-100 consecutive adenine nucleotides.
  • the non-adenine nucleotide is after one, two, three, four, five, six, or seven adenine nucleotides and is followed by at least 8 consecutive adenine nucleotides.
  • the poly-A tail may comprise one sequence of consecutive adenine nucleotides followed by one or more non-adenine nucleotides, optionally followed by additional adenine nucleotides.
  • the poly-A tail comprises or contains one non-adenine nucleotide or one consecutive stretch of 2-10 non-adenine nucleotides.
  • the non-adenine nucleotide(s) is located after at least 8, 9, 10, 11, or 12 consecutive adenine nucleotides.
  • the one or more non-adenine nucleotides are located after at least 8-50 consecutive adenine nucleotides.
  • the one or more non-adenine nucleotides are located after at least 8, 9, 10, 11, 12, 13, 14, 15. 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38. 39, 40, 41, 42, 43, 41, 45, 46, 47, 48, 49, or 50 consecutive adenine nucleotides.
  • the non-adenine nucleotide is guanine, cytosine, or thymine. In some instances, the non-adenine nucleotide is a guanine nucleotide. In some embodiments, the non-adenine nucleotide is a cytosine nucleotide. In some embodiments, the non-adenine nucleotide is a thymine nucleotide.
  • the non-adenine nucleotide may be selected from: a) guanine and thymine nucleotides; b) guanine and cytosine nucleotides; c) thymine and cytosine nucleotides; or d) guanine, thymine and cytosine nucleotides.
  • An exemplary poly-A tail comprising non-adenine nucleotides is provided as SEQ ID NO: 62.
  • the mRNA is purified.
  • the mRNA is purified using a precipation method (e.g., LiCl precipitation, alcohol precipitation, or an equivalent method, e.g., as described herein).
  • the mRNA is purified using a chromatography-based method, such as an HPLC-based method or an equivalent method (e.g., as described herein),
  • the mRNA is purified using both a precipitation method (e.g., LiCl precipitation) and an HPLC-based method.
  • At least one gRNA is provided in combination with an mRNA disclosed herein.
  • a gRNA is provided as a separate molecule from the mRNA.
  • a gRNA is provided as a part, such as a part of a UTR, of an mRNA disclosed herein.
  • the gRNA is chemically modified.
  • a gRNA comprising one or more modified nucleosides or nucleotides is called a “modified” gRNA or “chemically modified” gRNA, to describe the presence of one or more non-naturally and/or naturally occurring components or configurations that are used instead of or in addition to the canonical A, G, C, and U residues.
  • a modified gRNA is synthesized with a non-canonical nucleoside or nucleotide, is here called “modified.”
  • Modified nucleosides and nucleotides can include one or more of: (i) alteration, e.g., replacement, of one or both of the non-linking phosphate oxygens and/or of one or more of the linking phosphate oxygens in the phosphodiester backbone linkage (an exemplary backbone modification); (ii) alteration, e.g., replacement, of a constituent of the ribose sugar, e.g., of the 2′ hydroxyl on the ribose sugar (an exemplary sugar modification); (iii) wholesale replacement of the phosphate moiety with “dephospho” linkers (an exemplary backbone modification); (iv) modification or replacement of a naturally occurring nucleobase, including with a non-canonical nucleobase (an exemplary base modification); (v) replacement or modification of the rib
  • a gRNA comprises a modified uridine at some or all uridine positions.
  • the modified uridine is a uridine modified at the 5 position, e.g., with a halogen or C1-C6 alkoxy.
  • the modified undine is a pseudouridine modified at the I position, e.g., with a C1-C6 alkyl.
  • the modified uridine can be, for example, pseudouridine, N1-methyl-pseudouridine, 5-methoxyuridine, 5-iodouridine, or a combination thereof.
  • the modified uridine is 5-methoxy-oridine.
  • the modified uridine is 5-iodouridine.
  • the modified uridine is pseudouridine. In some embodiments the modified uridine is N1-methyl-pseudouridine. In some embodiments, the modified uridine is a combination of pseudouridine and N1-methyl-pseudouridine. In some embodiments, the modified uridine is a combination of pseudouridine and 5-methoxyuridine. In some embodiments, the modified uridine is a combination of N1-methyl pseudouridine and 5-methoxyuridine. In some embodiments, the modified uridine is a combination of 5-iodouridine and N1-methyl-pseudouridine. In some embodiments, the modified uridine is a combination of pseudouridine and 5-iodouridine. In some embodiments, the modified uridine is a combination of 5-iodouridine and 5-methoxyuridine.
  • At least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% of the uridine positions in a gRNA according to the disclosure are modified uridines.
  • 10%-25%, 15-25%, 25-35%, 35-45%, 45-55%, 55-65%, 65-75%, 75-85%, 85-95%, or 90-100% of the uridine positions in a gRNA according to the disclosure are modified uridines, e.g., 5-methoxyuridine, 5-iodouridine, NI-methyl pseudouridine, pseudouridine, or a combination thereof.
  • 10%-25%, 15-25%, 25-35%, 35-45%, 45-55%, 55-65%, 65-75%, 75-85%, 85-95%, or 90-100% of the uridine positions in a gRNA according to the disclosure are 5-methoxyuridine. In some embodiments, 10%-25%, 15-25%, 25-35%, 35-45%, 45-55%, 55-65%, 65-75%, 75-85%, 85-95%, or 90-100% of the uridine positions in a gRNA according to the disclosure are pseudouridine.
  • 10%-25%, 15-25%, 25-35%, 35-45%, 45-55%, 55-65%, 65-75%, 75-85%, 85-95%, or 90-100% of the uridine positions in a gRNA according to the disclosure are N1-methyl pseudouridine. In some embodiments, 10%-25%, 15-25%, 25-35%, 35-45%, 45-55%, 55-65%, 65-75%, 75-85%, 85-95%, or 90-100% of the uridine positions in a gRNA according to the disclosure are 5-iodouridine.
  • 10%-25%, 15-25%, 25-35%, 35-45%, 45-55%, 55-65%, 65-75%, 75-85%, 85-95%, or 90-100% of the uridine positions in a gRNA according to the disclosure are 5-methoxyuridine, and the remainder are N1-methyl pseudouridine.
  • 10%-25%, 15-25%, 25-35%, 35-45%, 45-55%, 55-65%, 65-75%, 75-85%, 85-95%, or 90-100% of the uridine positions in a gRNA according to the disclosure are 5-iodouridine, and the remainder are N1-methyl pseudouridine.
  • modified gRNAs comprising nucleosides and nucleotides (collectively “residues”) that can have two, three, four, or more modifications.
  • a modified residue can have a modified sugar and a modified nucleobase.
  • every base of a gRNA is modified, e.g., all bases have a modified phosphate group, such as a phosphorothioate group.
  • all, or substantially all, of the phosphate groups of an gRNA molecule are replaced with phosphorothioate groups.
  • modified gRNAs comprise at least one modified residue at or near the 5′ end of the RNA.
  • modified gRNAs comprise at least one modified residue at or near the 3′ end of the RNA.
  • the gRNA comprises one, two, three or more modified residues.
  • at least 5% e.g., at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%. at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100%
  • modified nucleosides or nucleotides are modified nucleosides or nucleotides.
  • Unmodified nucleic acids can be prone to degradation by, e.g., intracellular nucleases or those found in serum.
  • nucleases can hydrolyze nucleic acid phosphodiester bonds.
  • the gRNAs described herein can contain one or more modified nucleosides or nucleotides, e.g., to introduce stability toward intracellular or serum-based nucleases.
  • the modified gRNA molecules described herein can exhibit a reduced innate immune response when introduced into a population of cells, both in vivo and ex vivo.
  • innate immune response includes a cellular response to exogenous nucleic acids, including single stranded nucleic acids, which involves the induction of cytokine expression and release, particularly the interferons, and cell death.
  • the phosphate group of a modified residue can be modified by replacing one or more of the oxygens with a different substituent.
  • the modified residue e.g., modified residue present in a modified nucleic acid
  • the backbone modification of the phosphate backbone can include alterations that result in either an uncharged linker or a charged linker with unsymmetrical charge distribution.
  • modified phosphate groups include, phosphorothioate, phosphoroselenates, borano phosphates, borano phosphate esters, hydrogen phosphonates, phosphoroamidates, alkyl or aryl phosphonates and phosphotriesters.
  • the phosphorous atom in an unmodified phosphate group is achiral. However, replacement of one of the non-bridging oxygens with one of the above atoms or groups of atoms can render the phosphorous atom chiral.
  • the stereogenic phosphorous atom can possess either the configuration (herein Rp) or the “S” configuration (herein Sp).
  • the backbone can also be modified by replacement of a bridging oxygen, (i.e., the oxygen that links the phosphate to the nucleoside), with nitrogen (bridged phosphoroamidates), sulfur (bridged phosphorothioates) and carbon (bridged methylenephosphonates).
  • a bridging oxygen i.e., the oxygen that links the phosphate to the nucleoside
  • nitrogen bridged phosphoroamidates
  • sulfur bridged phosphorothioates
  • carbon bridged methylenephosphonates
  • the phosphate group can be replaced by non-phosphorus containing connectors in certain backbone modifications.
  • the charged phosphate group can be replaced by a neutral moiety.
  • moieties which can replace the phosphate group can include, without limitation, e.g., methyl phosphonate, hydroxylamino, siloxane, carbonate, carboxymethyl, carbamate, amide, thioether, ethylene oxide linker, sulfonate, sulfonamide, thioformacetal, formacetal, oxime, methyleneimino, methylenemethylimino, methylenehydrazo, methylenedimethylhydrazo and methyleneoxymethylimino.
  • compositions and methods disclosed herein may include a template nucleic acid.
  • the template may be used to alter or insert a nucleic acid sequence at or near a target site for a Cas nuclease.
  • the methods comprise introducing a template to the cell.
  • a single template may be provided.
  • two or more templates may be provided such that editing may occur at two or more target sites.
  • different templates may be provided to edit a single gene in a cell, or two different genes in a cell.
  • the template may be used in homologous recombination. In some embodiments, the homologous recombination may result in the integration of the template sequence or a portion of the template sequence into the target nucleic acid molecule. In other embodiments, the template may be used in homology-directed repair, which involves DNA strand invasion at the site of the cleavage in the nucleic acid. In some embodiments, the homology-directed repair may result in including the template sequence in the edited target nucleic acid molecule. In yet other embodiments, the template may be used in gene editing mediated by non-homologous end joining. In some embodiments, the template sequence has no similarity to the nucleic acid sequence near the cleavage site. In some embodiments, the template or a portion of the template sequence is incorporated. In some embodiments, the template includes flanking inverted terminal repeat (ITR) sequences.
  • ITR flanking inverted terminal repeat
  • the template may comprise a first homology arm and a second homology arm (also called a first and second nucleotide sequence) that are complementary to sequences located upstream and downstream of the cleavage site, respectively.
  • a first homology arm and a second homology arm also called a first and second nucleotide sequence
  • each arm can be the same length or different lengths, and the sequence between the homology arms can be substantially similar or identical to the target sequence between the homology arms, or it can be entirely unrelated.
  • the degree of complementarity or percent identity between the first nucleotide sequence on the template and the sequence upstream of the cleavage site, and between the second nucleotide sequence on the template and the sequence downstream of the cleavage site may permit homologous recombination, such as, e.g., high-fidelity homologous recombination, between the template and the target nucleic acid molecule.
  • the degree of complementarity may be about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100%. In some embodiments, the degree of complementarity may be about 95%, 97%, 98%, 99%, or 100%.
  • the degree of complementarity may be at least 98%, 99%, or 100%. In some embodiments, the degree of complementarity may be 100%. In some embodiments, the percent identity may be about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100%. In some embodiments, the percent identity may be about 95%, 97%, 98%, 99%, or 100%. In some embodiments, the percent identity may be at least 98%, 99%, or 100%. In some embodiments, the percent identity may be 100%.
  • the template sequence may correspond to, comprise, or consist of an endogenous sequence of a target cell. It may also or alternatively correspond to, comprise, or consist of an exogenous sequence of a target cell.
  • endogenous sequence refers to a sequence that is native to the cell.
  • exogenous sequence refers to a sequence that is not native to a cell, or a sequence whose native location in the genome of the cell is in a different location.
  • the endogenous sequence may be a genomic sequence of the cell.
  • the endogenous sequence may be a chromosomal or extrachromosomal sequence.
  • the endogenous sequence may be a plasmid sequence of the cell.
  • the template sequence may be substantially identical to a portion of the endogenous sequence in a cell at or near the cleavage site, but comprise at least one nucleotide change.
  • editing the cleaved target nucleic acid molecule with the template may result in a mutation comprising an insertion, deletion, or substitution of one or more nucleotides of the target nucleic acid molecule,
  • the mutation may result in one or more amino acid changes in a protein expressed from a gene comprising the target sequence.
  • the mutation may result in one or more nucleotide changes in an RNA expressed from the target gene.
  • the mutation may alter the expression level of the target gene. In some embodiments, the mutation may result in increased or decreased expression of the target gene. in some embodiments, the mutation may result in gene knock-down. In some embodiments, the mutation may result in gene knock-out. In some embodiments, the mutation may result in restored gene function. In some embodiments, editing of the cleaved target nucleic acid molecule with the template may result in a change in an exon sequence, an intron sequence, a regulatory sequence, a transcriptional control sequence, a translational control sequence, a splicing site, or a non-coding sequence of the target nucleic acid molecule, such as DNA.
  • the template sequence may comprise an exogenous sequence.
  • the exogenous sequence may comprise a protein or RNA coding sequence operably linked to an exogenous promoter sequence such that, upon integration of the exogenous sequence into the target nucleic acid molecule, the cell is capable of expressing the protein or RNA encoded by the integrated sequence.
  • the expression of the integrated sequence may be regulated by an endogenous promoter sequence.
  • the exogenous sequence may provide a cDNA sequence encoding a protein or a portion of the protein.
  • the exogenous sequence may comprise or consist of an exon sequence, an intron sequence, a reaulatory sequence, a transcriptional control sequence, a translational control sequence, a splicing site, or a non-coding sequence.
  • the integration of the exogenous sequence may result in restored gene function.
  • the integration of the exogenous sequence may result in a gene knock-in.
  • the integration of the exogenous sequence may result in a gene knock-out.
  • the template may be of any suitable length.
  • the template may comprise 10, 15, 20, 25, 50, 75, 100, 150, 200, 500, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000, 5500, 6000, or more nucleotides in length.
  • the template may be a single-stranded nucleic acid.
  • the template can be double-stranded or partially double-stranded nucleic acid.
  • the single stranded template is 20, 30, 40, 50, 75, 100, 125, 150, 175, or 200 nucleotides in length.
  • the template may comprise a nucleotide sequence that is complementary to a portion of the target nucleic acid molecule comprising the target sequence (i.e., a “homology arm”).
  • the template may comprise a homology arm that is complementary to the sequence located upstream or downstream of the cleavage site on the target nucleic acid molecule.
  • the template contains ssDNA or dsDNA containing flanking invert-terminal repeat (ITR) sequences.
  • the template is provided as a vector, plasmid, minicircle, nanocircle, or PCR product.
  • the nucleic acid is purified. In some embodiments, the nucleic acid is purified using a precipation method (e.g., LiCl precipitation, alcohol precipitation, or an equivalent method, e.g., as described herein). In some embodiments, the nucleic acid is purified using a chromatography-based method, such as an HPLC-based method or an equivalent method (e.g., as described herein). In some embodiments, the nucleic is purified using both a precipitation method (e.g., LiCl precipitation) and an HPLC-based method.
  • a precipation method e.g., LiCl precipitation, alcohol precipitation, or an equivalent method, e.g., as described herein.
  • a chromatography-based method such as an HPLC-based method or an equivalent method (e.g., as described herein).
  • the nucleic is purified using both a precipitation method (e.g., LiCl precipitation) and an HPLC-based method
  • a CRISPR/Cas system of the present disclosure may be directed to and cleave a target sequence on a target nucleic acid molecule.
  • the target sequence may be recognized and cleaved by the Cas nuclease.
  • a target sequence for a Cas nuclease is located near the nuclease's cognate PAM sequence.
  • a Class 2 Cas nuclease may be directed by a gRNA to a target sequence of a target nucleic acid molecule, where the gRNA hybridizes with and the Class 2 Cas protein cleaves the target sequence.
  • the guide RNA hybridizes with and a Class 2 Cas nuclease cleaves the target sequence adjacent to or comprising its cognate PAM.
  • the target sequence may be complementary to the targeting sequence of the guide RNA.
  • the degree of complementarity between a targeting sequence of a guide RNA and the portion of the corresponding target sequence that hybridizes to the guide RNA may be about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100%.
  • the percent identity between a targeting sequence of a guide RNA and the portion of the corresponding target sequence that hybridizes to the guide RNA may be about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100%.
  • the homology region of the target is adjacent to a cognate PAM sequence.
  • the target sequence may comprise a sequence 100% complementary with the targeting sequence of the guide RNA.
  • the target sequence may comprise at least one mismatch, deletion, or insertion, as compared to the targeting sequence of the guide RNA.
  • the length of the target sequence may depend on the nuclease system used.
  • the targeting sequence of a guide RNA for a CRISPRICas system may comprise 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, or more than 50 nucleotides in length and the target sequence is a corresponding length, optionally adjacent to a PAM sequence.
  • the target sequence may comprise 15-24 nucleotides in length.
  • the target sequence may comprise 17-21 nucleotides in length.
  • the target sequence may comprise 20 nucleotides in length.
  • the target sequence may comprise a pair of target sequences recognized by a pair of nickases that cleave opposite strands of the DNA molecule. In some embodiments, the target sequence may comprise a pair of target sequences recognized by a pair of nickases that cleave the same strands of the DNA molecule. In some embodiments, the target sequence may comprise a part of target sequences recognized by one or more Cas nucleases.
  • the target nucleic acid molecule may be any DNA or RNA molecule that is endogenous or exogenous to a cell.
  • the target nucleic acid molecule may be an episomal DNA, a plasmid, a genomic DNA, viral genome, mitochondrial DNA, or chromosomal DNA from a cell or in the cell.
  • the target sequence of the target nucleic acid molecule may be a genomic sequence from a cell or in a cell, including a human cell.
  • the target sequence may be a viral sequence. In further embodiments, the target sequence may be a pathogen sequence. In yet other embodiments, the target sequence may be a synthesized sequence. In further embodiments, the target sequence may be a chromosomal sequence. In certain embodiments, the target sequence may comprise a translocation junction, e.g., a translocation associated with a cancer. In some embodiments, the target sequence may be on a eukaryotic chromosome, such as a human chromosome. In certain embodiments, the target sequence is a liver-specific sequence, in that it is expressed in liver cells.
  • the target sequence may be located in a coding sequence of a gene, an intron sequence of a gene, a regulatory sequence, a transcriptional control sequence of a gene, a translational control sequence of a gene, a splicing site or a non-coding sequence between genes.
  • the gene may be a protein coding gene.
  • the gene may be a non-coding RNA gene.
  • the target sequence may comprise all or a portion of a disease-associated gene.
  • the target sequence may be located in a non-genic functional site in the genome, for example a site that controls aspects of chromatin organization, such as a scaffold site or locus control region.
  • the target sequence may be adjacent to a protospacer adjacent motif (“PAM”).
  • PAM protospacer adjacent motif
  • the PAM may be adjacent to or within 1, 2, 3, or 4, nucleotides of the 3′ end of the target sequence.
  • the length and the sequence of the PAM may depend on the Cas protein used.
  • the PAM may be selected from a consensus or a particular PAM sequence for a specific Cas9 protein or Cas9 ortholog, including those disclosed in FIG. 1 of Ran et al., Nature, 520: 186-191 (2015), and FIG. S5 of Zetsche 2015, the relevant disclosure of each of which is incorporated herein by reference.
  • the PAM may be 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides in length.
  • Non-limiting exemplary PAM sequences include NGG, NGGNG, NG, NAAAAN, NNAAAAW, NNNNACA, GNNNCNNA, TTN, and NNNNGATT (wherein N is defined as any nucleotide, and W is defined as either A or T).
  • the PAM sequence may be NGG.
  • the PAM sequence may be NGGNG.
  • the PAM sequence may be TTN.
  • the PAM sequence may be NNAAAAW.
  • LNP formulations for RNAs including CRISPR/Cas camos.
  • Such LNP formulations include an “amine lipid”, along with a helper lipid, a neutral lipid, and a PEG lipid.
  • such LNP formulations include an “amine lipid”, along with a helper lipid and a PEG lipid.
  • the LNP formulations include less than 1 percent neutral phospholipid.
  • the LNP formulations include less than 0.5 percent neutral phospholipid.
  • lipid nanoparticle is meant a particle that comprises a plurality of (i.e. more than one) lipid molecules physically associated with each other by intermolecular forces.
  • the LNP compositions for the delivery of biologically active agents comprise an “amine lipid”, which is defined as Lipid A or its equivalents, including acetal analogs of Lipid A.
  • the amine lipid is Lipid A, which is (9Z,12Z)-3-((4,4-bis(octyloxy)butanoyl)oxy)-2-((((3-(diethylamino)propoxy)carbonyl)oxy)methyl) propyl octadeca-9,12-dienoate, also called 3-((4,4-bis(octyloxy)butanoyl)oxy)-2-(((3-(diethylamino)propoxy)carbonyl)oxy)methyl)propyl (9Z,12Z)-octadeca-9,12-dienoate.
  • Lipid A can be depicted as:
  • Lipid A may be synthesized according to WO2015/095340 (e.g., pp. 84-86).
  • the amine lipid is an equivalent to Lipid A.
  • an amine lipid is an analog of Lipid A.
  • a Lipid A analog is an acetal analog of Lipid A.
  • the acetal analog is a C4-C12 acetal analog.
  • the acetal analog is a C5-C12 acetal analog.
  • the acetal analog is a C5-C10 acetal analog.
  • the acetal analog is chosen from a C4, C5, C6, C7, C9, C10, C11, and C 12 acetal analog.
  • Amine lipids suitable for use in the LNPs described herein are biodegradable in vivo and suitable for delivering a biologically active avent, such as an RNA to a cell.
  • the amine lipids have low toxicity (e.g., are tolerated in an animal model without adverse effect in amounts of greater than or equal to 10 mg/kg of RNA cargo).
  • LNPs comprising an amine lipid include those where at least 75% of the amine lipid is cleared from the plasma within 8, 10, 12, 24, or 48 hours, or 3, 4, 5, 6, 7, or 10 days.
  • LNPs comprising an amine lipid include those where at least 50% of the mRNA or gRNA is cleared from the plasma within 8, 10, 12, 24, or 48 hours, or 3, 4, 5, 6, 7, or 10 days.
  • LNPs comprising an amine lipid include those where at least 50% of the LNP is cleared from the plasma within 8, 10, 12, 24, or 48 hours, or 3, 4, 5, 6, 7, or 10 days, for example by measuring a lipid (e.g., an amine lipid), RNA (e.g., mRNA), or another component.
  • lipid-encapsulated versus free lipid, RNA, or nucleic acid component of the LNP is measured.
  • Lipid clearance may be measured as described in literature. See Maier, M. A., et al. Biodegradable Lipids Enabling Rapidly Eliminated Lipid Nanoparticles for Systemic Delivery of RNAi Therapeutics. Mol. Tiler, 2013, 21(8), 1570-78 (“Maier”).
  • Maier LNP-siRNA systems containing luciferases-targeting siRNA were administered to six- to eight-week old male C57Bl/6 mice at 0.3 mg/kg by intravenous bolus injection via the lateral tail vein. Blood, liver, and spleen samples were collected at 0.083, 0.25, 0.5, 1, 2, 4, 8, 24, 48, 96, and 168 hours post-dose.
  • mice were perfused with saline before tissue collection and blood samples were processed to obtain plasma. All samples were processed and analyzed by LC-MS. Further, Maier describes a procedure for assessing toxicity after administration of LNP-siRNA formulations. For example, a luciferase-targeting siRNA was administered at 0, 1, 3, 5, and 10 mg/kg (5 animals/group) via single intravenous bolus injection at a dose volume of 5 mL/kg to male Sprague-Dawley rats. After 24 hours, about 1 mL of blood was obtained from the jugular vein of conscious animals and the serum was isolated. At 72 hours post-dose, all animals were euthanized for necropsy.
  • a luciferase-targeting siRNA was administered at 0, 1, 3, 5, and 10 mg/kg (5 animals/group) via single intravenous bolus injection at a dose volume of 5 mL/kg to male Sprague-Dawley rats. After 24 hours, about 1 mL of blood
  • the amine lipids may lead to an increased clearance rate.
  • the clearance rate is a lipid clearance rate, for example the rate at which a lipid is cleared from the blood, serum, or plasma.
  • the clearance rate is an RNA clearance rate, for example the rate at which an mRNA or a gRNA is cleared from the blood, serum, or plasma.
  • the clearance rate is the rate at which LNP is cleared from the blood, serum, or plasma.
  • the clearance rate is the rate at which LNP is cleared from a tissue, such as liver tissue or spleen tissue.
  • a high clearance rate leads to a safety profile with no substantial adverse effects.
  • the amine lipids may reduce LNP accumulation in circulation and in tissues. In some embodiments, a reduction in LNP accumulation in circulation and in tissues leads to a safety profile with no substantial adverse effects.
  • the amine lipids of the present disclosure are ionizable (e.g., may form a salt) depending upon the pH of the medium they are in.
  • the amine lipids may be protonated and thus bear a positive charge.
  • a slightly basic medium such as, for example, blood, where pH is approximately 7.35
  • the amine lipids may not be protonated and thus bear no charge.
  • the amine lipids of the present disclosure may be protonated at a pH of at least about 9.
  • the amine lipids of the present disclosure may be protonated at a pH of at least about 9.
  • the amine lipids of the present disclosure may be protonated at a pH of at least about 10.
  • the pH at which an amine lipid is predominantly protonated is related to its intrinsic pKa.
  • the amine lipids of the present disclosure may each, independently, have a pKa in the range of from about 5.1 to about 7.4. In some embodiments, the amine lipids of the present disclosure may each, independently, have a pKa in the range of from about 5.5 to about 6.6. In some embodiments, the amine lipids of the present disclosure may each, independently, have a pKa in the range of from about 5.6 to about 6.4. In some embodiments, the amine lipids of the present disclosure may each, independently, have a pKa in the range of from about 5.8 to about 6.2.
  • the amine lipids of the present disclosure may each, independently, have a pKa in the range of from about 5.8 to about 6.5.
  • the pKa of an amine lipid can be an important consideration in formulating LNPs as it has been found that cationic lipids with a pKa ranging from about 5.1 to about 7.4 are effective for delivery of cargo in vivo, e.g., to the liver. Furthermore, it has been found that cationic lipids with a pKa ranging from about 5.3 to about 6.4 are effective for delivery in vivo, e.g., to tumors. See, e.g., WO 2014/136086,
  • Neutral lipids suitable for use in a lipid composition of the disclosure include, for example, a variety of neutral, uncharged or zwitterionic lipids.
  • Examples of neutral phospholipids suitable for use in the present disclosure include, but are not limited to, 5-heptadecylbenzene-1,3-diol (resorcinol), dipalmitoylphosphatidylcholine (DPPC), distearoylphosphatidylcholine (DSPC), pohsphocholine (DOPC), dimyristoylphosphatidylcholine (DMPC), phosphatidylcholine (PLPC), 1,2-distearoyl-sn-glycero-3-phosphocholine (DAPC), phosphatidylethanolamine (PE), egg phosphatidylcholine (EPC), dilauryloylphosphatidylcholine (DLPC), dimyristoylphosphatidylcholine (DMPC), 1-myristoyl-2-pal
  • the neutral phospholipid may be selected from the group consisting of distearoylphosphatidylcholine (DSPC) and dimyristoyl phosphatidyl ethanolamine (DMPE).
  • the neutral phospholipid may be distearoylphosphatidylcholine (DSPC).
  • the neutral phospholipid may be dipahnitoylphosphatidylcholine (DPPC).
  • Helper lipids include steroids, sterols, and alkyl resorcinois.
  • Helper lipids suitable for use in the present disclosure include, but are not limited to, cholesterol, 5-heptadecylresorcinol, and cholesterol hemisuccinate,
  • the helper lipid may be cholesterol.
  • the helper lipid may be cholesterol hemisuccinate.
  • PEG lipids are stealth lipids that alter the length of time the nanoparticles can exist in vivo (e.g., in the blood). PEG lipids may assist in the formulation process by, for example, reducing particle aggregation and controlling particle size. PEG lipids used herein may modulate phannacokinetic properties of the LNPs. Typically, the PEG lipid comprises a lipid moiety and a polymer moiety based on PEG.
  • the lipid moiety may be derived from diacylglycerol or diacylglycamide, including those comprising a dialkylglycerol or dialkylglycamide group having alkyl chain length independently comprising from about C4 to about C40 saturated or unsaturated carbon atoms, wherein the chain may comprise one or more functional groups such as, for example, an amide or ester.
  • the alkyl chain length comprises about C10 to C20.
  • the dialkylglycerol or dialkylalyeamide group can further comprise one or more substituted alkyl groups.
  • the chain lengths may be symmetrical or assymetric.
  • PEG polyethylene glycol or other polyalkylene ether polymer.
  • PEG moiety is an optionally substituted linear or branched polymer of ethylene glycol or ethylene oxide.
  • PEG moiety is Alternatively, the PEG moiety may be substituted, e.g., by one or more alkyl, alkoxy, acyl, hydroxy, or aryl groups.
  • the PEG moiety includes PEG copolymer such as PEG-polyurethane or PEG-polypropylene (see, e.g., J.
  • the PEG moiety does not include PEG copolymers, e.g., it may be a PEG monopolymer.
  • the PEG has a molecular weight of from about 130 to about 50,000, in a sub-embodiment, about 150 to about 30,000, in a sub-embodiment, about 150 to about 20,000, in a sub-embodiment about 150 to about 15,000, in a sub-embodiment, about 150 to about 10,000, in a sub-embodiment, about 150 to about 6,000, in a sub-embodiment, about 150 to about 5,000, in a sub-embodiment, about 150 to about 4,000, in a sub-embodiment, about 150 to about 3,000, in a sub-embodiment, about 300 to about 3,000, in a sub-embodiment,
  • the PEG (e.g., conjugated to a lipid moiety or lipid, such as a stealth lipid), is a “PEG-2K,” also termed “PEG 2000,” which has an average molecular weight of about 2,000 daltons.
  • PEG-2K is represented herein by the following formula (I), wherein n is 45, meaning that the number averaged degree of polymerization comprises about 45 subunits
  • n may range from about 30 to about 60. In some embodiments, n may range from about 35 to about 55. In some embodiments, n may range from about 40 to about 50. In some embodiments, n may range from about 42 to about 48. In some embodiments, n may be 45.
  • R may be selected from H, substituted alkyl, and unsubstituted alkyl. In some embodiments, R may be unsubstituted alkyl. In some embodiments, R may be methyl.
  • the PEG lipid may be selected from PEG-dilauroylidycerol, PEG-dimyristoylglycerol (PEG-DMG) (catalog #GM-020 from NOF, Tokyo, Japan), PEG-dipalmitoylglycerol, PEG-distearoylglycerol (PEG-DSPE) (catalog #DSPE-020CN, NOF, Tokyo, Japan), PEG-dilaurylglycamide, PEG-dimyristylglycamide, PEG-dipalmitoylglyeamide, and PEG-distearoylglycamide, PEG-cholesterol (1-[8′-(Cholest-5-en-3[beta]-oxy)carboxamido-3′,6′-dioxaoctanyl]carbamoyl-[omega]-methyl-poly(ethylene glycol), PEG-DMB (3
  • the PEG lipid may be PEG2k-DMG. In some embodiments, the PEG lipid may be PEG2k-DSG. In one embodiment, the PEG lipid may be PEG2k-DSPE.
  • the PEG lipid may be PEG2k-DMA. In one embodiment, the PEG lipid may be PEG2k-C-DMA. In one embodiment, the PEG lipid may be compound S027, disclosed in WO2016/010840 at paragraphs [00240] to [00244]. In one embodiment, the PEG lipid may be PEG2k-DSA. En one embodiment, the PEG lipid may be PEG2k-C11. In some embodiments, the PEG lipid may be PEG2k-C14. In some embodiments, the PEG lipid may be PEG2k-C16. In some embodiments, the PEG lipid may be PEG2k-C18.
  • Embodiments of the present disclosure provide lipid compositions described according to the respective molar ratios of the component lipids in the formulation.
  • the mol-% of the amine lipid may be from about 30 mol-% to about 60 mol-%. In one embodiment, the mol-% of the amine lipid may be from about 40 mol-% to about 60 mol-%. In one embodiment, the mol-% of the amine lipid may be from about 45 mol-% to about 60 mol-%. In one embodiment, the mol-% of the amine lipid may be from about 50 mol-% to about 60 mol-%.
  • the mol-% of the amine lipid may be from about 55 mol-% to about 60 mol-%. In one embodiment, the mol-% of the amine lipid may be from about 50 mol-% to about 55 mol-%. In one embodiment, the mol-% of the amine lipid may be about 50 mol-%. In one embodiment, the mol-% of the amine lipid may be about 55 mol-%. In some embodiments, the amine lipid mol-% of the LNP batch will be ⁇ 30%, ⁇ 25%, ⁇ 20%, ⁇ 15%, ⁇ 10%, ⁇ 5%, or ⁇ 2.5% of the target mol-%.
  • the amine lipid mol-% of the LNP batch will be ⁇ 4 mol-%, ⁇ 3 mol-%, ⁇ 2 mol-%, ⁇ 1.5 mol-%, ⁇ 1 mol-%, ⁇ 0.5 mol-%, or ⁇ 0.25 mol-% of the target mol-%. All mol-% numbers are given as a fraction of the lipid component of the LNP compositions. In certain embodiments, LNP inter-lot variability of the amine lipid mol-% will be less than 15%, less than 10% or less than 5%.
  • the mol-% of the neutral lipid may be from about 5 mol-% to about 15 mol-%. In one embodiment, the mol-% of the neutral lipid, e.g., neutral phospholipid, may be from about 7 mol-% to about 12 mol-%. In one embodiment, the mol-% of the neutral lipid, e.g., neutral phospholipid, may be from about 0 mol-% to about 5 mol-%. In one embodiment, the mol-% of the neutral lipid, e.g., neutral phospholipid, may be from about 0 mol-% to about 10 mol-%.
  • the mol-% of the neutral lipid may be from about 5 mol-% to about 10 mol-%. In one embodiment, the mol-% of the neutral lipid, e.g., neutral phospholipid, may be from about 8 mol-% to about 10 mol-%.
  • the mol-% of the neutral lipid may be about 5 mol-%, about 6 mol-%, about 7 mol-%, about 8 mol-%, about 9 mol-%, about 10 mol-%, about 11 mol-%, about 12 mol-%, about 13 mol-%, about 14 mol-%, or about 15 mol-%. In one embodiment, the mol-% of the neutral lipid, e.g., neutral phospholipid, may be about 9 mol-%.
  • the mol-% of the neutral lipid may be from about 1 mol-% to about 5 mol-%. In one embodiment, the mol-% of the neutral lipid may be from about 0.1 mol-% to about 1 mol-%. In one embodiment, the mol-% of the neutral lipid such as neutral phospholipid may be about 0.1 mol-%, about 0.2 mol-%, about 0,5 mol-%, 1 mol-%, about 1.5 mol-%, about 2 mol-%, about 2.5 moi-%, about 3 mol-%, about 3.5 mol-%, about 4 mol-%, about 4.5 mol-%, or about 5 mol-%.
  • the mol-% of the neutral lipid may be less than about 1 mol-%. In one embodiment, the mol-% of the neutral lipid, neutral phospholipid, may be less than about 0.5 mol-%, In one embodiment, the mol-% of the neutral lipid, e.g., neutral phospholipid, may be about 0 mol-%, about 0.1 mol-%, about 0.2 mol-%, about 0.3 mol-%, about 0.4 mol-%, about 0.5 mol-%, about 0.6 mol-%, about 0.7 mol-%, about 0.8 mol-%, about 0.9 mol-%, or about 1 mol-%.
  • the formulations disclosed herein are free of neutral lipid (i.e., 0 mol-% neutral lipid). In some embodiments, the formulations disclosed herein are essentially free of neutral lipid (i.e., about 0 mol-% neutral lipid). In some embodiments, the formulations disclosed herein are free of neutral phospholipid (i.e., 0 mol-% neutral phospholipid). In some embodiments, the formulations disclosed herein are essentially free of neutral phospholipid (i.e., about 0 mol-% neutral phospholipid).
  • the neutral lipid mol-% of the LNP batch will be ⁇ 30%, ⁇ 25%, ⁇ 20%, ⁇ 10%, ⁇ 5%., or ⁇ 12.5% of the target neutral lipid mol-%.
  • LNP inter-lot variability will be less than 15%, less than 10% or less than 5%.
  • the mol-% of the helper lipid may be from about 20 mol-% to about 60 mol-%. In one embodiment, the mol-% of the helper lipid may be from about 25 mol-% to about 55 mol-%. In one embodiment, the mol-% of the helper lipid may be from about 25 mol-% to about 50 mol-%. In one embodiment, the mol-% of the helper lipid may be from about 25 mol-% to about 40 mol-%. In one embodiment, the mol-% of the helper lipid may he from about 30 mol-% to about 50 mol-%.
  • the mol-% of the helper lipid may be from about 30 mol-% to about 40 mol-%. In one embodiment, the mol-% of the helper lipid is adjusted based on amine lipid, neutral lipid, and PEG lipid concentrations to bring the lipid component to 100 mol-%. In one embodiment, the mol-% of the helper lipid is adjusted based on amine lipid and PEG lipid concentrations to bring the lipid component to 100 mol-%. In one embodiment, the mol-% of the helper lipid is adjusted based on amine lipid and PEG lipid concentrations to bring the lipid component to at least 99 mol-%.
  • the helper mol-% of the LNP batch will be ⁇ 30%, ⁇ 25%, ⁇ 20%, ⁇ 15%, ⁇ 10%, ⁇ 5%, or ⁇ 12.5% of the target mol-%.
  • LNP inter-lot variability will be less than 15%, less than 10% or less than 5%.
  • the mol-% of the PEG lipid may be from about 1 mol-% to about 10 mol-%. In one embodiment, the mol-% of the PEG lipid may be from about 2 mol-% to about 10 mol-%. In one embodiment, the mol-% of the PEG lipid may be from about 2 mol-% to about 8 mol-%. In one embodiment, the mol-% of the PEG lipid may be from about 2 mol-% to about 4 mol-%. In one embodiment, the mol-% of the PEG lipid may be from about 2.5 mol-% to about 4 mol-%. In one embodiment, the mol-% of the PEG lipid may be about 3 mol-%.
  • the mol.-% of the PEG lipid may be about 2.5 mol-%.
  • the PEG lipid mol-% of the LNP batch will be ⁇ 30%, ⁇ 25%, ⁇ 20%, ⁇ 15%, ⁇ 10%, ⁇ 5%, or ⁇ 12.5% of the target PEG lipid mol-%.
  • LNP inter-lot variability will be less than 15%, less than 10% or less than 5%.
  • the cargo includes an mRNA encoding an RNA-guided DNA-binding agent (e.g. a Cas nuclease, a Class 2 Cas nuclease, or Cas9), and a gRNA or a nucleic acid encoding a gRNA, or a combination of mRNA and gRNA.
  • an LNP composition may comprise a Lipid A or its equivalents.
  • the amine lipid is Lipid A.
  • the amine lipid is a Lipid A equivalent, e.g. an analog of Lipid A.
  • the amine lipid is an acetal analog of Lipid A.
  • an LNP composition comprises an amine lipid, a neutral lipid, a helper lipid, and a PEG lipid.
  • the helper lipid is cholesterol.
  • the neutral lipid is DSPC.
  • PEG lipid is PEG2k-DMG.
  • an LNP composition may comprise a Lipid A, a helper lipid, a neutral lipid, and a PEG lipid.
  • an LNP composition comprises an amine lipid, DSPC, cholesterol, and a PEG lipid.
  • the LNP composition comprises a PEG lipid comprising DMG.
  • the amine lipid is selected from Lipid A, and an equivalent of Lipid A, including an acetal analog of Lipid A.
  • an LNP composition comprises Lipid A, cholesterol, DSPC, and PEG2k-DMG.
  • an LNP composition comprises an amine lipid, a helper lipid, a neutral lipid, and a PEG lipid.
  • an LNP composition comprises an amine lipid, a helper lipid, a neutral phospholipid, and a PEG lipid.
  • an LNP composition comprises a lipid component that consists of an amine lipid, a helper lipid, a neutral lipid, and a PEG lipid.
  • an LNP composition comprises an amine lipid, a helper lipid, and a PEG lipid.
  • an LNP composition does not comprise a neutral lipid, such as a neutral phospholipid.
  • an LNP composition comprises a lipid component that consists of an amine lipid, a helper lipid, and a PEG lipid.
  • the neutral lipid is chosen from one or more of DSPC, DPPC, DAPC, DMPC, DOPC, DOPE, and DSPE.
  • the neutral lipid is DSPC.
  • the neutral lipid is DPPC.
  • the neutral lipid is DAPC.
  • the neutral lipid is DMPC.
  • the neutral lipid is DOPC.
  • the neutral lipid is DOPE.
  • the neutral lipid is DSPE.
  • the helper lipid is cholesterol.
  • the PEG lipid is PEG2k-DMG.
  • an LNP composition may comprise a Lipid A, a helper lipid, and a PEG lipid.
  • an LNP composition may comprise a lipid component that consists of Lipid A, a helper lipid, and a PEG lipid.
  • an LNP composition comprises an amine lipid, cholesterol, and a PEG lipid.
  • an LNP composition comprises a lipid component that consists of an amine lipid, cholesterol, and a PEG lipid.
  • the LNP composition comprises a PEG lipid comprising DMG.
  • the amine lipid is selected from Lipid A and an equivalent of Lipid A, including an acetal analog of Lipid A.
  • the amine lipid is a C5-C12 or a C4-C12 acetal analog of Lipid A.
  • an LNP composition comprises Lipid A, cholesterol, and PEG2k-DMG.
  • Embodiments of the present disclosure also provide lipid compositions described according to the molar ratio between the positively charged amine groups of the amine lipid (N) and the negatively charged phosphate groups (P) of the nucleic acid to be encapsulated. This may be mathematically represented by the equation N/P.
  • an LNP composition may comprise a lipid component that comprises an amine lipid, a helper lipid, a neutral lipid, and a PEG lipid; and a nucleic acid component, wherein the N/P ratio is about 3 to 10.
  • an LNP composition may comprise a lipid component that comprises an amine lipid, a helper lipid, and a PEG lipid; and a nucleic acid component, wherein the N/P ratio is about 3 to 10.
  • an LNP composition may comprise a lipid component that comprises an amine lipid, a helper lipid, a neutral lipid, and a helper lipid; and an RNA component, wherein the N/P ratio is about 3 to 10.
  • an LNP composition may comprise a lipid component that comprises an amine lipid, a helper lipid, and a PEG lipid; and an RNA component, wherein the N/P ratio is about 3 to 10.
  • the N/P ratio may be about 5 to 7. In one embodiment, the N/P ration may be about 3 to 7. In one embodiment, the N/P ratio may be about 4.5 to 8. In one embodiment, the N/P ratio may be about 6. In one embodiment, the N/P ratio may he 6 ⁇ 1. In one embodiment, the N/P ratio may be 6 ⁇ 0.5. In some embodiments, the N/P ratio will be ⁇ 30%, ⁇ 25%, ⁇ 20%, ⁇ 15%, ⁇ 10%, ⁇ 5%, or ⁇ 2.5% of the target N/P ratio. In certain embodiments, LNP inter-lot variability will be less than 15%. less than 10% or less than 5%.
  • the nucleic acid component may comprise an mRNA, such as an snRNA encoding a Cas nuclease.
  • An RNA component includes RNA, optionally with additional nucleic acid and/or protein, e.g., RNP cargo.
  • RNA comprises a Cas9 mRNA.
  • the LNP further comprises a gRNA nucleic acid, such as a gRNA.
  • the RNA component comprises a Cas nuclease mRNA and a gRNA.
  • the RNA component comprises a Class 2 Cas nuclease mRNA and a sRNA.
  • an LNP composition may comprise an mRNA encoding a Cas nuclease such as a Class 2 Cas nuclease, an amine lipid, a helper lipid, a neutral lipid, and a PEG lipid.
  • an LNP composition may comprise an mRNA encoding a Cas nuclease such as a Class 2 Cas nuclease, an amine lipid, a helper lipid, and a PEG lipid.
  • the helper lipid is cholesterol
  • the neutral lipid is DSPC.
  • the PEG lipid is PEG2k-DMG or PEG2k-C11.
  • compositions comprising an mRNA encoding a Cas nuclease such as a Class 2 Cas nuclease
  • the amine lipid is selected from Lipid A and its equivalents, such as an acetal analog of Lipid A.
  • an LNP composition may comprise a gRNA.
  • an LNP composition may comprise an amine lipid, a gRNA, a helper lipid, a neutral lipid, and a PEG lipid.
  • an LNP composition may comprise an amine lipid, a gRNA, a helper lipid, and a PEG lipid.
  • the helper lipid is cholesterol.
  • the neutral lipid is DSPC.
  • the PEG lipid is PEG2k-DMG or PEG2k-C11.
  • the amine lipid is selected from Lipid A and its equivalents, such as an acetal analog of Lipid A.
  • an LNP composition may comprise an sgRNA. In one embodiment, an LNP composition may comprise a Cas9 sgRNA. In one embodiment, an LNP composition may comprise a Cpf1 sgRNA. In some compositions comprising an sgRNA, the LNP includes an amine lipid, a helper lipid, a neutral lipid, and a PEG lipid. In some compositions comprising an sgRNA, the LNP includes an amine lipid, a helper lipid, and a PEG lipid. In certain compositions comprising an sgRNA, the helper lipid is cholesterol. In other compositions comprising an sgRNA, the neutral lipid is DSPC.
  • the PEG lipid is PEG2k-DMG or PEG2k-C11.
  • the amine lipid is selected from Lipid A and its equivalents, such as acetal analogs of Lipid A.
  • an LNP composition comprises an mRNA encoding a Cas nuclease and a gRNA, which may be an sgRNA.
  • an LNP composition may comprise an amine lipid, an mRNA encoding a Cas nuclease, a gRNA, a helper lipid, a neutral lipid, and a PEG lipid.
  • an LNP composition may comprise a lipid component consisting of an amine lipid, a helper lipid, a neutral lipid, and a PEG lipid; and a nucleic acid component consisting of an mRNA encoding a Cas nuclease, and a gRNA.
  • an LNP composition may comprise a lipid component consisting of an amine lipid, a helper lipid, and a PEG lipid; and a nucleic acid component consisting of an mRNA encoding a Cas nuclease, and a gRNA.
  • the helper lipid is cholesterol.
  • the neutral lipid is DSPC.
  • compositions comprising an mRNA encoding a Cas nuclease and a gRNA comprise less than about 1 mol-% neutral lipid, e.g. neutral phospholipid.
  • Certain compositions comprising an mRNA encoding a Cas nuclease and a gRNA comprise less than about 0.5 mol-% neutral lipid, e.g. neutral phospholipid.
  • the LNP does not comprise a neutral lipid, e.g., neutral phospholipid.
  • the PEG lipid is PEG2k-DMG or PEG2k-C11.
  • the amine lipid is selected from Lipid A and its equivalents, such as acetal analogs of Lipid A.
  • the LNP compositions include a Cas nuclease mRNA, such as a Class 2 Cas mRNA and at least one gRNA.
  • the LNP composition includes a ratio of gRNA to Gas nuclease mRNA, such as Class 2 Gas nuclease mRNA from about 25:1 to about 1:25,
  • the LNP formulation includes a ratio of gRNA to Cas nuclease mRNA, such as Class 2 Cas nuclease mRNA from about 10:1 to about 1:10.
  • the LNP formulation includes a ratio of gRNA to Cas nuclease mRNA, such as Class 2 Cas nuclease mRNA from about 8:1 to about 1:8. As measured herein, the ratios are by weight. In some embodiments, the LNP formulation includes a ratio of gRNA to Cas nuclease mRNA, such as Class 2 Cas mRNA from about 5:1 to about 1:5. In some embodiments, ratio range is about 3:1 to 1:3, about 2:1 to 1:2, about 5:1 to 1:2, about 5:1 to 1:1, about 3:1 to 1:2, about 3:1 to 1:1, about 3:1, about 2:1 to 1:1.
  • the gRNA to mRNA ratio is about 3:1 or about 2:1 In some embodiments the ratio of gRNA to Cas nuclease mRNA, such as Class 2 Cas nuclease is about 1:1. The ratio may be about 25:1, 10:1, 5:1, 3:1, 1:1, 1:3, 1:5, 1:0, or 1:25.
  • the LNP compositions disclosed herein may include a template nucleic acid.
  • the template nucleic acid may be co-formulated with an mRNA encoding a Cas nuclease, such as a Class 2 Cas nuclease mRNA.
  • the template nucleic acid may be co-formulated with a guide RNA.
  • the template nucleic acid may be co-formulated with both an mRNA encoding a Cas nuclease and a guide RNA.
  • the template nucleic acid may be formulated separately from an mRNA encoding a Cas nuclease or a guide RNA.
  • the template nucleic acid may be delivered with, or separately from the LNP compositions.
  • the template nucleic acid may be single- or double-stranded, depending on the desired repair mechanism.
  • the template may have regions of homology to the target DNA, or to sequences adjacent to the target DNA.
  • LNPs are formed by mixing an aqueous RNA solution with an organic solvent-based lipid solution, e.g., 100% ethanol.
  • Suitable solutions or solvents include or may contain: water, PBS, Iris buffer, NaCl, citrate buffer, ethanol, chloroform, diethylether, cyclohexane, tetrahydrofuran, methanol, isopropanol.
  • a pharmaceutically acceptable buffer e.g., for in vivo administration of LNPs, may be used.
  • a buffer is used to maintain the pH of the composition comprising LNPs at or above pH 6.5.
  • a buffer is used to maintain the pH of the composition comprising LNPs at or above pH 7.0.
  • the composition has a pH ranging from about 7.2 to about 7.7.
  • the composition has a pH ranging from about 7.3 to about 7.7 or ranging from about 7.4 to about 7.6.
  • the composition has a pH of about 7.2, 7.3, 7.4, 7.5, 7.6, or 7.7.
  • the pH of a composition may be measured with a micro pH probe.
  • a cryoprotectant is included in the composition.
  • cryoprotectants include sucrose, trehalose, glycerol, DMSO, and ethylene glycol.
  • Exemplary compositions may include up to 10% cryoprotectant, such as, for example, sucrose.
  • the LNP composition may include about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10% cryoprotectant.
  • the LNP composition may include about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10% sucrose.
  • the LNP composition may include a buffer.
  • the buffer may comprise a phosphate buffer (PBS), a Tris buffer, a citrate buffer, or mixtures thereof.
  • the buffer comprises NaCl. In certain emboidments, NaCl is omitted. Exemplary amounts of NaCl may range from about 20 mM to about 45 mM.
  • Exemplary amounts of NaCl may range from about 40 mM to about 50 mM. In some embodiments, the amount of NaCl is about 45 mM.
  • the buffer is a Tris buffer. Exemplary amounts of Tris may range from about 20 mM to about 60 mM. Exemplary amounts of Tris may range from about 40 mM to about 60 mM. In some embodiments, the amount of Tris is about 50 mM.
  • the buffer comprises NaCl and Tris. Certain exemplary embodiments of the LNP compositions contain 5% sucrose and 45 mM NaCl in Tris buffer.
  • compositions contain sucrose in an amount of about 5% w/v, about 45 mM NaCl, and about 50 mM Tris at pH 7.5.
  • the salt, buffer, and cryoprotectant amounts may be varied such that the osmolality of the overall formulation is maintained.
  • the final osmolality may he maintained at less than 450 mOsm/L.
  • the osmolality is between 350 and 250 mOsm/L.
  • Certain embodiments have a final osmolality of 300+/ ⁇ 20 mOsm/L.
  • inicrofluidic mixing, T-mixing, or cross-mixing is used.
  • flow rates, junction size, junction geometry, junction shape, tube diameter, solutions, and/or RNA and lipid concentrations may be varied.
  • LNPs or LNP compositions may be concentrated or purified, e.g., via dialysis, tangential flow filtration, or chromatography.
  • the LNPs may be stored as a suspension, an emulsion, or a lyophilized powder, for example.
  • an LNP composition is stored at 2-8° C., in certain aspects, the LNP compositions are stored at room temperature.
  • an LNP composition is stored frozen, for example at ⁇ 20′ C or ⁇ 80° C.
  • an LNP composition is stored at a temperature ranging from about 0° C. to about ⁇ 80° C. Frozen LNP compositions may be thawed before use, for example on ice, at room temperature, or at 25° C.
  • the LNPs may be, e.g., microspheres (including unilamellar and multilamellar vesicles, e.g., “liposomes”—lamellar phase lipid bilayers that, in some embodiments, are substantially spherical—and, in more particular embodiments, can comprise an aqueous core, e.g., comprising a substantial portion of RNA molecules), a dispersed phase in an emulsion, micelles, or an internal phase in a suspension.
  • microspheres including unilamellar and multilamellar vesicles, e.g., “liposomes”—lamellar phase lipid bilayers that, in some embodiments, are substantially spherical—and, in more particular embodiments, can comprise an aqueous core, e.g., comprising a substantial portion of RNA molecules), a dispersed phase in an emulsion, micelles, or an internal phase in a suspension.
  • the LNP compositions are biodegradable, in that they do not accumulate to cytotoxic levels in vivo at a therapeutically effective dose. In some embodiments, the LNP compositions do not cause an innate immune response that leads to substantial adverse effects at a therapeutic dose level. In some embodiments, the LNP compositions provided herein do not cause toxicity at a therapeutic dose level.
  • the pdi may range from about 0.005 to about 0.75. In some embodiments, the pdi may range from about 0.01 to about 0.5. In some embodiments, the pdi may range from about zero to about 0.4. In some embodiments, the pdi may range from about zero to about 0.35. In some embodiments, the pdi may range from about zero to about 0.35. In some embodiments, the pdi may range from about zero to about 0.3. In some embodiments, the pdi may range from about zero to about 0.25. In some embodiments, the pdi may range from about zero to about 0.2. In some embodiments, the pdi may be less than about 0.08, 0.1, 0.15, 0.2, or 0.4.
  • the LNPs disclosed herein have a size (e.g., Z-average diameter) of about 1 to about 250 nm. In some embodiments, the LNPs have a size of about 10 to about 200 nm. In further embodiments, the LNPs have a size of about 20 to about 150 nm. In some embodiments, the LNPs have a size of about 50 to about 150 nm. In some embodiments, the LNPs have a size of about 50 to about 100 nm. In some embodiments, the LNPs have a size of about 50 to about 120 nm. In some embodiments, the LNPs have a size of about 60 to about 100 nm.
  • the LNPs have a size of about 75 to about 150 nm. In some embodiments, the LNPs have a size of about 75 to about 120 nm. In some embodiments, the LNPs have a size of about 75 to about 100 nm. Unless indicated otherwise, all sizes referred to herein are the average sizes (diameters) of the fully formed nanoparticles, as measured by dynamic light scattering on a Malvern Zetasizer. The nanoparticle sample is diluted in phosphate buffered saline (PBS) so that the count rate is approximately 200-400 kcps. The data is presented as a weighted-average of the intensity measure (Z-average diameter).
  • PBS phosphate buffered saline
  • the LNPs are formed with an average encapsulation efficiency ranging from about 50% to about 100%. In some embodiments, the LNPs are formed with an average encapsulation efficiency ranging from about 50% to about 70%. In some embodiments, the LNPs are formed with an average encapsulation efficiency ranging from about 70% to about 90%. In some embodiments, the LNPs are formed with an average encapsulation efficiency ranging from about 90% to about 100%. In some embodiments, the LNPs are formed with an average encapsulation efficiency ranging from about 75% to about 95%.
  • the LNPs are formed with an average molecular weight ranging from about 1.00E+05 g/mol to about 1.00E+10 g/mol. In some embodiments, the LNPs are formed with an average molecular weight ranging from about 5.00E+05 g/mol to about 7.00E+07 g/mol. In some embodiments, the LNPs are formed with an average molecular weight ranging from about 1.00E+06 g/mol to about 1.00E+10 g/mol. In some embodiments, the LNPs are formed with an average molecular weight ranging from about 1.00E+07 g/mol to about 1.00E+09 g/mol. In some embodiments, the LNPs are formed with an average molecular weight ranging from about 5.00E+06 g/mol to about 5.00E+09 g/mol.
  • the polydispersity (Mw/Mn; the ratio of the weight averaged molar mass (Mw) to the number averaged molar mass (Mn)) may range from about 1.000 to about 2.000. In some embodiments, the Mw/Mn may range from about 1.00 to about 1.500. In some embodiments, the Mw/Mn may range from about 1.020 to about 1.400. In some embodiments, the Mw/Mn may range from about 1.010 to about 1.100. In some embodiments, the Mw/Mn may range from about 1.100 to about 1.350.
  • the LNP compositions disclosed herein may be used in methods for engineering cells through gene editing, both in vivo and in vitro. In some embodiments, the methods involve contacting a cell with an LNP composition described herein.
  • methods involve contacting a cell in a subject, such as a mammal, such as a human.
  • the cell is in an organ, such as a liver, such as a mammalian liver, such as a human liver.
  • the cell is a liver cell, such as a mammalian liver cell, such as a human liver cell.
  • the cell is a hepatocyte, such as a mammalian hepatocyte, such as a human hepatocyte.
  • the liver cell is a stem cell.
  • the human liver cell may be a liver sinusoidal endothelial cell (LSEC).
  • the human liver cell may be a Kupffer cell. In some embodiments, the human liver cell may be a hepatic stellate cell. In some embodiments, the human liver cell may be a tumor cell. In some embodiments, the human liver cell may be a liver stem cell. In additional embodiments, the cell comprises ApoE-binding receptors. In some embodiments, the liver cell such as a hepatocyte is in situ. In some embodiments, the Jiver cell such as a hepatocyte is isolated, e.g., in a culture, such as in a primary culture. Also provided are methods corresponding to the uses disclosed herein, which comprise administering the LNP compositions disclosed herein to a subject or contacting a cell such as those described above with the LNP compositions disclosed herein
  • engineered cells are provided, for example an engineered cell derived from any one of the cell types in the preceding paragraph. Such engineered cells are produced according to the methods described herein. In some embodiments, the engineered cell resides within a tissue or organ, e.g., a liver within a subject.
  • a cell comprises a modification, for example an insertion or deletion (“indel”) or substitution of nucleotides in a target sequence.
  • the modification comprises an insertion of 1, 2, 3, 4 or 5 or more nucleotides in a target sequence.
  • the modification comprises an insertion of either 1 or 2 nucleotides in a target sequence.
  • the modification comprises a deletion of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20 or 25 or more nucleotides in a target sequence.
  • the modification comprises a deletion of either 1 or 2 nucleotides in a target sequence.
  • the modification comprises an indel which results in a frameshift mutation in a target sequence. In some embodiments, the modification comprises a substitution of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20 or 25 or more nucleotides in a target sequence. In some embodiments, the modification comprises a substitution of either 1 or 2 nucleotides in a target sequence. In some embodiments, the modification comprises one or more of an insertion, deletion, or substitution of nucleotides resulting from the incorporation of a template nucleic acid, for example any of the template nucleic acids described herein.
  • a population of cells comprising engineered cells is provided, for example a population of cells comprising cells engineered according to the methods described herein.
  • the population comprises engineered cells cultured in vitro.
  • the population resides within a tissue or organ, e.g., a liver within a subject.
  • at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90% or at least 95% or more of the cells within the population is engineered.
  • a method disclosed herein results in at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90% or at least 95% editing efficiency (or “percent editing”), defined by detetion of indels.
  • a method disclosed herein results in at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90% or at least 95% DNA modification efficiency, defined by detecting a change in sequence, whether by insertion, deletion, substitution or otherwise.
  • a method disclosed herein results in an editing efficiency level or a DNA modification efficiency level of between about 5% to about 100%, about 10% to about 50%, about 20 to about 100%, about 20 to about 80%, about 40 to about 100%, or about 40 to about 80% in a cell population.
  • cells within the population comprise a modification, e.g., an indel or substitution at a target sequence.
  • the modification comprises an insertion of 1, 2, 3, 4 or 5 or more nucleotides in a target sequence, In some embodiments, the modification comprises an insertion of either 1 or 2 nucleotides in a target sequence. In other embodiments, the modification comprises a deletion of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20 or 25 or more nucleotides in a target sequence. In some embodiments, the modification comprises a deletion of either 1 or 2 nucleotides in a target sequence. In some embodiments, the modification results in a frameshift mutation in a target sequence.
  • the modification comprises an indel which results in a frameshift mutation in a target sequence. In some embodiments, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% or more of the engineered cells in the population comprise a frameshift mutation. In some embodiments, the modification comprises a substitution of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20 or 25 or more nucleotides in a target sequence. In some embodiments, the modification comprises a substitution of either 1 or 2 nucleotides in a target sequence. In some embodiments, the modification comprises one or more of an insertion, deletion, or substitution of nucleotides resulting from the incorporation of a template nucleic acid, for example any of the template nucleic acids described herein.
  • the LNP compositions disclosed herein may be used for gene editing in vivo and in vitro.
  • one or more LNP compositions described herein may be administered to a subject in need thereof.
  • one or more LNP compositions described herein may contact a cell.
  • a therapeutically effective amount of a composition described herein may contact a cell of a subject in need thereof.
  • a genetically engineered cell may be produced by contacting a cell with an LNP composition described herein.
  • the methods comprise introducing a template nucleic acid to a cell or subject, as set forth above.
  • the methods involve administering the LNP composition to a cell associated with a liver disorder. In some embodiments, the methods involve treating a liver disorder. In certain embodiments, the methods involve contacting a hepatic cell with the LNP composition. In certain embodiments, the methods involve contacting a hepatocyte with the LNP composition. In some embodiments, the methods involve contacting an ApoE binding cell with the LNP composition.
  • an LNP composition comprising an mRNA encoding a Class 2 Cas nuclease and a gRNA may be administered to a cell, such as an ApoE binding cell.
  • a template nucleic acid is also introduced to the cell.
  • an LNP composition comprising, a Class 2 Cas nuclease and an sgRNA may be administered to a cell, such as an ApoE binding cell.
  • an LNP composition comprising an mRNA encoding a Class 2 Cas nuclease, a gRNA, and a template may be administered to a cell.
  • an LNP composition comprising a Cas nuclease and an sgRNA may be administered to a liver cell. In some cases, the liver cell is in a subject.
  • a subject may receive a single dose of an LNP composition. In other examples, a subject may receive multiple doses of an LNP composition. In some embodiments, the LNP composition is administered 2-5 times. Where more than one dose is administered, the doses may be administered about 1, 2, 3, 4, 5, 6, 7, 14, 21, or 28 days apart; about 2, 3, 4, 5, or 6 months apart; or about 1, 2, 3, 4, or 5 years apart. In certain embodiments, editing improves upon readministration of an LNP composition.
  • an LNP composition comprising an mRNA encoding a Cas nuclease such as a Class 2 Cas nuclease, may be administered to a cell, separately from the administration of a composition comprising a gRNA.
  • an LNP composition comprising an mRNA encoding a Cas nuclease such as a Class 2 Cas nuclease and a gRNA may be administered to a cell, separately from the administration of a template nucleic acid to the cell.
  • an LNP composition comprising an mRNA encoding a Cas nuclease such as a Class 2 Cas nuclease may be administered to a cell, followed by the sequential administration of an LNP composition comprising a gRNA and then a template to the cell.
  • an LNP composition comprising an mRNA encoding a Cas nuclease is administered before an LNP composition comprising a gRNA, the administrations may be separated by about 4, 6, 8, 12, or 24 hours; or 2, 3, 4, 5, 6, or 7 days.
  • the LNP compositions may be used to edit a gene resulting in a gene knockout. In an embodiment, the LNP compositions may be used to edit a gene resulting in gene knockdown in a population of cells. In another embodiment, the LNP compositions may be used to edit a gene resulting in a gene correction. In a further embodiment, the LNP compositions may be used to edit a cell resulting in gene insertion.
  • administration of the LNP compositions may result in gene editing which results in persistent response.
  • administration may result in a duration of response of a day, a month, a year, or longer.
  • “duration of response” means that, after cells have been edited using an LNP composition disclosed herein, the resulting modification is still present for a certain period of time after administration of the LNP composition.
  • the modification may be detected by measuring target protein levels.
  • the modification may be detected by detecting the target DNA.
  • the duration of response may be at least 1 week. In other embodiments, the duration of response may be at least 2 weeks. In one embodiment, the duration of response may be at least 1 month. In some embodiments, the duration of response may be at least 2 months.
  • the duration of response may be at least 4 months. In one embodiment, the duration of response may be at least 6 months. In certain embodiments, the duration of response may be about 26 weeks. In some embodiments, the duration of response may be at least 1 year. In some embodiments, the duration of response may be at least 5 years. In some embodiments, the duration of response may be at least 10 years. In some embodiments, a persistent response is detectable after at least 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 15, 18, 21, or 24 months, either by measuring target protein levels or by detection of the target DNA. In some embodiments, a persistent response is detectable after at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, or 20 years, either by measuring target protein levels or by detection of the target DNA.
  • the LNP compositions can be administered parenterally.
  • the LNP compositions may be administered directly into the blood stream, into tissue, into muscle, or into an internal organ. Administration may be systemic, e.g., to injection or infusion. Administration may be local. Suitable means for administration include intravenous, intraarterial, intrathecal, intraventricular, intraurethral, intrasternal, intracranial, subretinal, intravitreal, intra-anterior chamber, intramuscular, intrasynovial, intradermal, and subcutaneous.
  • Suitable devices for administration include needle (including microneedle) injectors, needle-free injectors, osmotic pumps, and infusion techniques.
  • the LNP compositions will generally, but not necessarily, be administered as a formulation in association with one or more pharmaceutically acceptable excipients.
  • excipient includes any ingredient other than the compound(s) of the disclosure, the other lipid component(s) and the biologically active agent.
  • An excipient may impart either a functional (e.g. drug release rate controlling) and/or a non-functional (e.g. processing aid or diluent) characteristic to the formulations.
  • a functional e.g. drug release rate controlling
  • a non-functional e.g. processing aid or diluent
  • the choice of excipient will to a large extent depend on factors such as the particular mode of administration, the effect of the excipient on solubility and stability, and the nature of the dosage form.
  • Parenteral formulations are typically aqueous or oily solutions or suspensions. Where the formulation is aqueous, excipients such as sugars (including but not restricted to glucose, mannitol, sorbitol, etc.) salts, carbohydrates and buffering agents (preferably to a pH of from 3 to 9), but, for some applications, they may be more suitably formulated with a sterile non-aqueous solution or as a dried form to be used in conjunction with a suitable vehicle such as sterile, pyrogen-free water (WFI).
  • excipients such as sugars (including but not restricted to glucose, mannitol, sorbitol, etc.) salts, carbohydrates and buffering agents (preferably to a pH of from 3 to 9), but, for some applications, they may be more suitably formulated with a sterile non-aqueous solution or as a dried form to be used in conjunction with a suitable vehicle such as sterile, pyrogen-free water (WFI).
  • WFI ster
  • Numeric ranges are inclusive of the numbers defining the range. Measured and measureable values are understood to be approximate, taking into account significant digits and the error associated with the measurement.
  • the term “about” or “approximately” means an acceptable error for a particular value as determined by one of ordinary skill in the art, which depends in part on how the value is measured or determined.
  • the use of a modifier such as “about” before a range or before a list of values, modifies each endpoint of the range or each value in the list. “About” also includes the value or enpoint. For example, “about 50-55” encompasses “about 50 to about 55”.
  • the use of “comprise”, “comprises”, “comprising”, “contain”, “contains”, “containing”, “include”, “includes”, and “including” is not limiting.
  • LNP formulations are identified on the X-axis based on their Lipid A mol-% and N:P ratios, labeled “% CL; N:P”.
  • PEG-2k-DMG concentrations of 2, 2.5, 3, 4, or 5 mol-% were formulated with (1) 45 mol-%, Lipid A; 4.5 N:P (“45; 4.5”); (2) 45 mol-% Lipid A; 6 N:P (“45; 6”); (3) 50 mol-% Lipid A; 4.5 N:P (“50; 4.5”); (4) 50 mol-% Lipid A; 6 N:P (“50; 6”); (5) 55 mol-% Lipid A; 4.5 N:P (“55; 4.5”); and (6) 55 mol-% Lipid A; 6 N:P (“55; 6”).
  • the DSPC mol-% was kept constant at 9 mol-% and the cholesterol mol-% was added to bring the balance of each formulation lipid component to 100 mol-%.
  • Each of the 30 formulations was formulated as described below, and administered as single dose at 1 mg per kg or 0.5 mg per kg doses of total RNA, ( FIG. 1 A and FIG. 1 B , respectively).
  • the lipid nanoparticle components were dissolved in 100% ethanol with the lipid component molar ratios set forth above.
  • the RNA cargos were dissolved in 25 mM citrate, 100 mM NaCl, pH 5.0, resulting in a concentration of RNA cargo of approximately 0.45 mg/mL.
  • the LNPs were formulated with a lipid amine to RNA phosphate (N:P) molar ratio of about 4.5 or about 6, with the ratio of mRNA to gRNA at 1:1 by weight.
  • the LNPs were formed by microfluidic mixing of the lipid and RNA solutions using a Precision Nanosystems NanoAssemblrTM Benchtop Instrument, according to the manufacturer's protocol. A 2:1 ratio of aqueous to organic solvent was maintained during mixing using differential flow rates. After mixing, the LNPs were collected, diluted in water (approximately 1:1 v/v), held for 1 hour at room temperature, and further diluted with water (approximately 1:1 v/v) before final buffer exchange. The final buffer exchange into 50 mM Tris, 45 mM NaCl, 5% (w/v) sucrose, pH 7.5 (TSS) was completed with PD-10 desalting columns (GE).
  • TSS pH 7.5
  • formulations were concentrated by centrifugation with Amicon 100 kDa centrifugal filters (Millipore). The resulting mixture was then filtered using a 0.2 ⁇ m sterile filter. The final LNP was stored at ⁇ 80° C. until further use.
  • DLS Dynamic Light Scattering
  • pdi polydispersity index
  • PDI Polydispersity index
  • Electropheretic light scattering is used to characterize the surface charge of the LNP at a specified pH.
  • the surface charge, or the zeta potential is a measure of the magnitude of electrostatic repulsion/attraction between particles in the LNP suspension.
  • Assymetric-Flow Field Flow Fractionation—Multi-Angle Light Scattering (AF4-MALS) is used to separate particles in the formulation by hydrodynamic radius and then measure the molecular weights, hydrodynamic radii and root mean square radii of the fractionated particles.
  • This allows the ability to assess molecular weight and size distributions as well as secondary characteristics such as the Burchard-Stockmeyer Plot (ratio of root mean square (“rms”) radius to hydrodynamic radius over time suggesting the internal core density of a particle) and the rms conformation plot (log of rms radius versus log of molecular weight where the slope of the resulting linear fit gives a degree of compactness versus elongation).
  • Nanoparticle tracking analysis (NTA, Malvern Nanosight) can be used to determine formulation particle size distribution as well as particle concentration. LNP samples are diluted appropriately and injected onto a microscope slide. A camera records the scattered light as the particles are slowly infused through field of view. After the movie is captured, the Nanoparticle Tracking Analysis processes the movie by tracking pixels and calculating a diffusion coefficient. This diffusion coefficient can be translated into the hydrodynamic radius of the particle. The instrument also counts the number of individual particles counted in the analysis to give particle concentration.
  • Cryo-electron microscopy (“cryo-EM”) can be used to determine the particle size, morphology, and structural characteristics of an LNP.
  • Lipid compositional analysis of the LNPs can be determined from liquid chromotography followed by charged aerosol detection (LC-CAD). This analysis can provide a comparison of the actual lipid content versus the theoretical lipid content.
  • LC-CAD charged aerosol detection
  • LNP formulations are analyzed for average particle size, polydispersity index (pdi), total RNA content, encapsulation efficiency of RNA, and zeta potential. LNP formualtions may be further characterized by lipid analysis, AF4-MALS, NTA, and/or cryo-EM. Average particle size and polydispersity are measured by dynamic light scattering (DLS) using a Malvern Zetasizer DLS instrument. LNP samples were diluted 30 ⁇ in PBS prior to being measured by DLS. Z-average diameter which is an intensity-based measurement of average particle size was reported along with number average diameter and pdi. A Malvern Zetasizer instrument is also used to measure the zeta potential of the LNP. Samples are diluted 1:17 (50 ⁇ L into 800 ⁇ L) in 0.1 ⁇ PBS, pH 7.4 prior to measurement.
  • a fluorescence-based assay (Ribogreen®, ThermoFisher Scientific) is used to determine total RNA concentration and free RNA. Encapsulation efficiency is calclulated as (Total RNA ⁇ Free RNA)/Total RNA.
  • LNP samples are diluted appropriately with 1 ⁇ TE buffer containing 0.2% Triton-X 100 to determine total RNA or 1 ⁇ TE buffer to determine free RNA. Standard curves are prepared by utilizing the starting RNA solution used to make the formulations and diluted in 1 ⁇ TE buffer+/ ⁇ 0.2% Triton-X 100.
  • Diluted RiboGreen® dye (according to the manufacturer's instructions) is then added to each of the standards and samples and allowed to incubate for approximately 10 minutes at room temperature, in the absence of light.
  • a SpectraMax M5 Microplate Reader (Molecular Devices) is used to read the samples with excitation, auto cutoff and emission wavelengths set to 488 nm, 515 nm, and 525 nm respectively. Total RNA and free RNA are determined from the appropriate standard curves.
  • Encapsulation efficiency is calclulated as (Total RNA ⁇ Free RNA)/Total RNA. The same procedure may be used for determining the encapsulation efficiency of a DNA-based or nucleic acid-containing cargo component.
  • Oligreen Dye may be used, and for double-strand DNA, Picogreen Dye.
  • AF4-MALS is used to look at molecular weight and size distributions as well as secondary statistics from those calculations.
  • LNPs are diluted as appropriate and injected into an AF4 separation channel using an HPLC autosampler where they are focused and then eluted with an exponential gradient in cross flow across the channel. All fluid is driven by an HPLC pump and Wyatt Eclipse Instrument. Particles eluting from the AF4 channel flow through a UV detector, multi-angle light scattering detector, quasi-elastic light scattering detector and differential refractive index detector.
  • Raw data is processed by using a Debeye model to determine molecular weight and rms radius from the detector signals.
  • Lipid components in LNPs are analyzed quantitatively by HPLC coupled to a charged aerosol detector (CAD). Chromatographic separation of 4 lipid components is achieved by reverse phase HPLC. CAD is a destructive mass-based detector which detects all non-volatile compounds and the signal is consistent regardless of analyte structure.
  • the Cas9 mRNA cargo was prepared by in vitro transcription.
  • Capped and polyadenylated Cas9 mRNA comprising 1 ⁇ NLS (SEQ ID NO:48) was generated by in vitro transcription using a linearized plasmid DNA template and T7 RNA polymerase.
  • Plasmid DNA containing a T7 promoter and a 100 nt poly(A/T) region was linearized by incubating at 37° C. for 2 hrs with XbaI with the following conditions: 200 ng/ ⁇ L plasmid. 2 U/ ⁇ L XbaI (NEB), and 1 ⁇ reaction buffer.
  • the XbaI was inactivated by heating the reaction at 65° C. for 20 min.
  • the linearized plasmid was purified from enzyme and buffer salts using a silica maxi spin column (Epoch Life Sciences) and analyzed by agarose gel to confirm linearization.
  • the IVT reaction to generate Cas9 modified mRNA was incubated at 37° C. for 4 hours in the following conditions: 50 ng/ ⁇ L linearized plasmid; 2 mM each of GTP, ATP, CTP, and N1-methyl pseudo-UTP (Trilink); 10 mM ARCA (Trilink); 5 U/ ⁇ L T7 RNA polymerase (NEB); 1 U/ ⁇ L Murine RNase inhibitor (NEB); 0.004 U/ ⁇ L Inorganic E. coli pyrophosphatase (NEB); and 1 ⁇ reaction buffer.
  • TURBO DNase ThermoFisher
  • ThermoFisher was added to a final concentration of 0.01 U/ ⁇ L, and the reaction was incubated for an additional 30 minutes to remove the DNA template.
  • the Cas9 mRNA was purified from enzyme and nucleotides using a MegaClear Transcription Clean-up kit per the manufacturer's protocol (ThermoFisher). Alternatively, the Cas9 mRNA was purified with a LiCl precipitation method.
  • the sgRNA in this example was chemically synthesized and sourced from a commercial supplier.
  • the final LNPs were characterized to determine the encapsulation efficiency, polydispersity index, and average particle size according to the analytical methods provided above.
  • mice single dose at 1 mg/kg or 0.5 mg/kg
  • genomic DNA was isolated for NGS analysis as described below.
  • CD-1 female mice ranging from 6 to 10 weeks of age were used in each study. Animals were weighed and grouped according to body weight for preparing dosing solutions based on group average weight. LNPs were dosed via the lateral tail vein in a volume of 0.2 mL per animal (approximately 10 mL per kilogram body weight). The animals were observed at approximately 6 hours post dose for adverse effects. Body weight was measured at twenty-four hours post-administration, and animals were euthanized at various time points by exsanguination via cardiac puncture under isoflurane anesthesia. Blood was collected into serum separator tubes or into tubes containing buffered sodium citrate for plasma as described herein. For studies involving in vivo editing, liver tissue was collected from the median lobe or from three independent lobes (e.g., the right median, left median, and left lateral lobes) from each animal for DNA extraction and analysis.
  • LNPs were dosed via the lateral tail vein in a volume of 0.2 mL per animal (
  • mice were measured for liver editing by Next-Generation Sequencing (NGS) and serum TTR levels (data not shown).
  • NGS Next-Generation Sequencing
  • TTR Transthyretin
  • mice TTR serum levels were determined using a Mouse Prealbumin (Transthyretin) ELISA Kit (Aviva Systems Biology, Cat. OKIA00111). Rat TTR serum levels were measured using a rat specific ELISA kit (Aviva Systems Biology catalog number OKIA00159) according to manufacture's protocol. Briefly, sera were serial diluted with kit sample diluent to a final dilution of 10,000-fold. This diluted sample was then added to the ELISA plates and the assay was then carried out according to directions.
  • kit sample diluent to a final dilution of 10,000-fold.
  • genomic DNA was isolated and deep sequencing was utilized to identify the presence of insertions and deletions introduced by gene editing.
  • PCR primers were designed around the target site (e.g., TTR), and the genomic area of interest was amplified. Primer sequences are provided below. Additional PCR was performed according to the manufacturer's protocols (Illumina) to add the necessary chemistry for sequencing. The amplicons were sequenced on an Illumina MiSeq instrument. The reads were aligned to the human reference genome (e.g., hg38) after eliminating those having low quality scores. The resulting files containing the reads were mapped to the reference genome (BAM files), where reads that overlapped the target region of interest were selected and the number of wild type reads versus the number of reads which contain an insertion, substitution, or deletion was calculated.
  • BAM files reference genome
  • the editing percentage (e.g., the “editing efficiency” or “percent editing”) is defined as the total number of sequence reads with insertions or deletions over the total number of sequence reads, including wild type.
  • FIG. 1 shows editing percentages in mouse liver as measured by NGS.
  • FIG. 1 A when 1 mg per kg RNA is dosed, in vivo editing percentages range from about 20% to over 60% liver editing.
  • FIG. 1 B At a 0.5 mg per kg dose, FIG. 1 B , about 10% to 60% liver editing was observed.
  • all compositions effectively delivered Cas9 mRNA and gRNA to the liver cells, with evidence of active CRISPR/Cas nuclease activity at the target site measured by NGS for each LNP composition.
  • LNPs containing 5% PEG lipid had lower encapsulation (data not shown), and somewhat reduced potency.
  • the LNPs were formed by impinging jet mixing of the lipid in ethanol with two volumes of RNA solutions and one volume of water.
  • the lipid in ethanol is mixed through a mixing cross with the two volumes of RNA solution.
  • a fourth stream of water is mixed with the outlet stream of the cross through an inline tee. (See WO2016010840 at FIG. 2 .)
  • the LNPs were maintained at room temperature for 1 hour, and then further diluted with water (approximately 1:1 v/v).
  • Diluted LNPs were concentrated using tangential flow filtration on a flat sheet cartridge (Sartorius, 100 kD MWCO) and then buffer exchanged by diafiltration into 50 mM Tris, 45 mM NaCl, 5% (w/v) sucrose, pH 7.5 (TSS). Alternatively, the final buffer exchange into TSS was completed with PD-10 desalting columns (GE). If required, formulations were concentrated by centrifugation with Amicon 100 kDa centrifugal filters (Millipore). The resulting mixture was then filtered using a 0.2 ⁇ m sterile filter. The final LNP was stored at 4° C. or ⁇ 80° C. until further use.
  • Cas9 mRNA and sgRNA were prepared as in Example 1, except that capped and poly-adenylated Cas9 U-depleted (Cas9 Udep) mRNA comprises SEQ ID N:43.
  • Sg282 is described in Example 1, and the sequence for sg534 (“G534”) is provided below:
  • LMP formulations were analyzed for average particle size, polydispersity (pdi), total RNA content and encapsulation efficiency of RNA as described in Example 1.
  • RNA analysis demonstrated the actual mol-% lipid levels, as indicated in Table 5 below.
  • Lipid Ratio Lipid A/Chol/ Lipid A Chol DSPC PEG DSPC/PEG mg/mL mg/mL mg/mL LNP (theoretical (theoretical (theoretical (theoretical ID # and actual) and actual) and actual) and actual) and actual) and actual) LNP898 45/44/9/2 18.0 8.0 3.3 2.3 46.1/42.6/9.2/2 18.3 7.7 3.4 2.4 LNP897 45/43/9/3 18.0 7.8 3.3 3.5 44.8/42.9/9.2/3.1 17.8 7.7 3.4 3.6 LNP966 50/38/9/3 33.4 11.5 5.6 5.8 50.0/38.0/8.8/3.1 35.6 12.3 5.8 6.5 LNP969 55/33/9/3 33.4 9.1 5.1 5.3 54.8/33.2/8.8/3.2 31.6 8.7 4.7 5.4
  • LNP897, LNP898, LNP966, and LNP969 were subjected to Asymmetric-Flow Field Flow Fractionation—Multi-Angle Light Scattering (AF4-MALS) analysis.
  • AF4-MALS Asymmetric-Flow Field Flow Fractionation—Multi-Angle Light Scattering
  • LNPs are injected into an AF4 separation channel using an HPLC autosampler where they are focused and then eluted with an exponential gradient in cross flow across the channel. All fluid is driven by an HPLC pump and Wyatt Eclipse Instrument. Particles eluting from the AF4 channel flow through a UV detector, Wyatt Heleos II multi-angle light scattering detector, quasi-elastic light scattering detector and Wyatt Optilab T-rEX differential refractive index detector. Raw data is processed in Wyatt Astra 7 Software by using a Debeye model to determine molecular weight and cats radius from the detector signals.
  • FIG. 2 A A log differential molar mass plot for the LNPs is provided as FIG. 2 A .
  • the X-axis indicates molar mass (g/mol), and the Y-axis indicates the differential number fraction.
  • the log differential molar mass plot shows the distribution of the different molecular weights measured for a specific formulation. This gives data towards the mode of the molecular weights as well as the overall distribution of molecular weights within the formulation, which gives a better picture of particle heterogeniety than average molecular weight.
  • the heterogeniety of the different LNP formulations are determined by measuring the different molar mass moments and calculating the ratio of the weight averaged molar mass (Mw) to the number averaged molar mass (Mn) to give a polydispersity of Mw/Mn.
  • the graph of the polydispersity for these different formulations is provided in FIG. 2 B .
  • the data indicate tighter particle distributions with 3 mol-% PEG, and with 50 and 55 mol-% Lipid A at N/P 6.0 as shown in FIG. 2 A . This is reflected in a tight polydispersity as shown in FIG. 2 B
  • the LNPs were formed as described in Example 2.
  • Cas9 mRNA and sgRNA were prepared as described above.
  • the LNPs compositions were characterized to determine the encapsulation efficiency, polydispersity index, and average particle size as described in Example 1.
  • lipid analysis demonstrated the actual mol-% lipid levels, as indicated in Table 8, below.
  • Lipid Ratio Lipid A/Chol/ Lipid A Chol. DSPC PEG DSPC/PEG mg/mL mg/mL mg/mL mg/mL LNP (theoretical (theoretical (theoretical (theoretical ID # and actual) and actual) and actual) and actual) and actual) and actual) LNP1021 50/38/9/3 23.6 8.1 3.9 4.1 50.9/37.4/8.6/3.1 21.6 7.2.
  • LNP1021, LNP1022, LNP1023, LNP1024 and LNP1025 were subjected to Asymmetric-Flow Field Flow Fractionation—Multi-Angle Light Scattering (AF4-MALS) analysis.
  • AF4-MALS Asymmetric-Flow Field Flow Fractionation—Multi-Angle Light Scattering
  • LNPs were run on AF4-MALS as described in Example 1.
  • FIG. 3 A A log differential molar mass plot for the LNPs is provided as FIG. 3 A .
  • the X-axis indicates molar mass (g/mol), and the Y-axis indicates the differential number fraction.
  • the log differential molar mass plot shows the distribution of the different molecular weights calculated for a specific formulation. This gives data towards the mode of the molecular weights as well as the overall distribution of molecular weights within the formulation, which gives a better picture of particle heterogeniety than average molecular weight.
  • Average molecular weight is plotted in FIG. 3 B .
  • the average molecular weight is the average of the entire distribution but gives no information about the shape of that distribution.
  • LNP1022 and LNP1025 have the same average molecular weight but LNP1022 has a slightly broader distribution.
  • the heterogeniety of the different LNP formulations are calculated by look at the different molar mass moments and calculating the ratio of the weight averaged molar mass (Mw) to the number averaged molar mass (Mn) to give a polydispersity of Mw/Mn.
  • the graph of the polydispersity for these different formulations is provided in FIG. 4 A .
  • FIG. 4 B a Burchard-Stockmeyer plot of the LNP formulations is provided as FIG. 4 B .
  • the Burchard-Stockmeyer plot shows the ratio of the rms radius versus the hydrodynamic radius across the elution of the formulation from the AF4 channel. This gives information towards the internal density of a lipid nanoparticle.
  • FIG. 4 B shows that LNP1021, LNP1022 and LNP1023 have different profiles in this measurement.
  • PEG DMG lipid was compared in LNP formulations comprising 2 mol-% or 3 mol-% of the PEG lipid. Compositons that comprise either 2 mol-%, or 3 mol-%, PEG DMG are provided in Table 9 below.
  • the LNPs were formed by the process described in Example 2.
  • G390 sg390
  • LNP formulations were analyzed for average particle size, polydispersity (pdi), total RNA content and encapsulation efficiency of RNA as described in Example 1.
  • lipid analysis demonstrated the actual mol-% lipid levels, as indicated in Table 11: below.
  • Lipid Ratio Lipid A/Chol/ Lipid A Chol. DSPC PEG DSPC/PEG mg/mL mg/mL mg/mL LNP (theoretical (theoretical (theoretical (theoretical ID # and actual) and actual) and actual) and actual) and actual) and actual) LNP809 45/44/9/2 28.6 12.7 5.3 3.7 45.7/43.3/9.0/2.1 30.5 13.1 5.6 4.0 LNP810 45/43/9/3 25.2 10.9 4.7 4.9 45.0/42.3/9.7/3.0 24.7 10.5 4.9 4.7
  • Rat serum cytokines were evaluated using a Luminex magnetic bead multiplex assay (Milliplex MAP magnetic bead assay from Millipore Sigma, catalog number RECYTMAG-65K) analyzing MCP-1, IL-6, TNF-alpha and IFN-gamma.
  • the assay beads were read on the BioRad BioPlex-200 and cytokine concentrations calculated off a standard curve using 4 parameter logistic fit with BioPlex Manager Software version 6.1. Data is graphed in FIG. 5 . See FIG. 5 A (serum TTR), FIG. 5 B (liver editing), and FIG. 5 C (cytokine p MCP1).
  • Rat TTR serum levels were measured using a rat specific ELISA kit (Aviva Systems Biology catalog number OKIA00159) according to manufacture's protocol. Briefly, serums were serially diluted with kit sample diluent to a final dilution of 10,000-fold. This diluted sample was then added to the ELISA plates and the assay was then carried out according to directions.
  • Genomic DNA was isolated from approximately 10 mg of liver tissue and analyzed using NGS as described above. PCR primer sequences for amplification are described below.
  • FIG. 5 A and FIG. 5 B show that serum TTR knockdown and liver editing were sufficient in the 2 mol-% and 3 mol-% PEG formulations.
  • FIG. 5 C shows that MCP-1 response is reduced using 3 mol-% PEG formulations.
  • LNP formulations prepared as described in Example 1 The particular molar amounts and cargos are provided in Tables 12-26. Each formulation containing Cas9 mRNA and guide RNA (gRNA) had a mRNA:gRNA ratio of 1:1 by weight. Doses of LNP (in mg/kg, total RNA content), route of administration and whether animals received pre-treatment of dexamethasone are indicated in the Tables. For animals receiving dexamethasone (Dex) pre-treatment, Dex was administered at 2 mg/kg by IV bolus injection, 1 hour prior to LNP or vehicle administration.
  • DEx dexamethasone
  • Kit reagents and standards were prepared as directed in the manufacturer's protocol. NHP serum was used neat. The plates were run on an MSD Sector Imager 6000 with analysis performed with MSD Discovery work bench software Version 4012.
  • Complement levels were measured in pre- and post-treated animals by enzyme Immunoassay.
  • Whole blood 0.5 mL was collected from a peripheral vein of restrained, conscious animals into a tube containing 0.5 mL k 2 EDTA. Blood was centrifuged at 2000 ⁇ g for 15 minutes. Plasma was aliquoted into 2 polypropylene microtubes of 120 ⁇ L each and stored at ⁇ 60 to ⁇ 86° C. until analysis.
  • a Quidel MicroVue Complement Plus EIA kit C3a-Cat #A031) or (Bb-Cat #A027) was used for analysis. Kit reagents and standards were prepared as directed in the manufacturer's protocol. The plates were run on an MSD Sector Imager 6000 at optical density at 450 nm. The results were analyzed using a 4-parameter curve fit.
  • Lipid 1 (DMG-PEG2k; Nof), is depicted as:
  • Lipid 2 synthesized as described in Heyes, et al., J. Controlled Release, 107 (2005), pp, 278-279 (See “Synthesis of PEG2000-C-DMA”), can be depicted as:
  • Lipid A was formulated with each PEG lipid at 2 mol-% and 3 mol-%.
  • the lipid nanoparticle components were dissolved in 100% ethanol with the lipid component molar ratios set forth above.
  • the RNA cargos were prepared in 25 mM citrate, 100 mM NaCl, pH 5.0, resulting in a concentration of RNA cargo of approximately 0.45 mg/mL.
  • the LNPs were formulated with a lipid amine to RNA phosphate (N:P) molar ratio of about 4.5 with the ratio of mRNA to gRNA at 1:1 by weight.
  • Cas9 mRNA, sg282, and LNPs were prepared as described in Example 1.
  • LNP compositions with Lipid 1, Lipid 2, or Lipid 3 were administered to female CD-1 mice and assessed as described in Example 1 at 1 mg/kg and 0.5 mg/kg of the body weight. Cohorts of mice were measured for liver editing by Next-Generation Sequencing (NGS) and serum TTR levels according to the methods of Example 1.
  • NGS Next-Generation Sequencing
  • FIG. 6 A and FIG. 6 B compare serum TTR levels between PEG lipid formulations.
  • FIG. 6 A shows serum TTR in ⁇ g/mL
  • FIG. 6 B shows the data as a percent knockdown (% TSS).
  • FIG. 6 C shows percent editing achieved in the liver.
  • the data indicate that LNP compositions with each of the tested PEG lipids tested potency at 2 mol-% and 3 mol-%, with Lipid 1 consistently performing slightly better than Lipid 2 and Lipid 3.
  • Lipid A A number of structural analogs of Lipid A were synthesized and tested in the LNP compositions described herein.
  • Lipid A is made by reacting 4,4-bis(octyloxy)butanoic acid (“Intermediate 13b” in Example 13 of WO2015/095340) with (9Z,12Z)-3-hydroxy-2-(hydroxymethyl)propyl octadeca-9,12-dienoate (“Intermediate 13c”), prior to addition of the head group by reacting the product of Intermediate 13b and Intermediate 13c with 3-diethylamino-1-propanol. (See pp. 84-86 of WO2015/095340.)
  • the C7, C9, and C10 analogs were formulated at 45 mol-% Lipid A Analog, 2 mol-% DMG-PEG2k, 9 mol-% DSPC, and 44 mol-% cholesterol, with an N:P ratio of 4.5.
  • Each analog was also formulated at 55 mol-% Lipid A Analog, 2.5 mol-% DMG-PEG2k, 9 mol-% DSPC, and 38.5 mol-% cholesterol, with an NT ratio of 6.
  • the lipid nanoparticle components were dissolved in 100% ethanol with the lipid component molar ratios set forth above.
  • the RNA cargos were prepared in 25 mM citrate, 100 mM NaCl, pH 5.0, resulting in a concentration of RNA cargo of approximately 0.45 mg/mL.
  • the RNA cargo included Cas9 mRNA comprising SEQ ID NO:43 and sg282, prepared as described above.
  • the LNPs were formed as described in Example 1.
  • the analogs were assessed for pKa using 6-(p-toluidino)-6-napthalene sulfonic acid (“TNS”) dissolved in water.
  • TNS 6-(p-toluidino)-6-napthalene sulfonic acid
  • 0.1 M phosphate buffer was prepared at different pH values ranging from 4.5 to 10.5.
  • Each analog was individually prepared in 100% ethanol, The lipid and TNS were then added in individual pH buffer and transferred to a plate to analyze at 321-488 nm wavelength on the SpectraMax plate reader. Values were plotted to generate pKa, log IC 50 is used as pKa.
  • mice Female CD-1 mice were dosed as described in Example 1 with 0.3 mg/kg ( FIG. 7 A - FIG. 7 E ), or with 0.1 mg per kg ( FIG. 7 F - FIG. 7 G ),
  • serum was collected for TTR analysis and liver was collected for editing analysis.
  • Serum TTR and percent editing assays were performed as described in Example 1.
  • the serum TTR levels and liver editing from FIG. 7 A - FIG. 7 E indicate that all the analogs performed comparably to Lipid A at 0.3 milligrams per kilogram body weight.
  • FIG. 7 F - FIG. 7 G indicate that while Lipid A had the highest potency, the newly synthesized analogs all have suitable TTR knockdown and liver editing.
  • PCH Primary cynomolgus liver hepatocytes.
  • PCH Primary cynomolgus liver hepatocytes (PCH) (Gibco) were thawed and resuspended in hepatocyte thawing medium with supplements (Gibco, Cat. CM7000) followed by centrifugation at 80 g for 4 minutes. The supernatant was discarded and the pelleted cells resuspended in hepatocyte plating medium plus supplement pack (Invitrogen, Cat. A1217601 and CM3000). Cells were counted and plated on Bio-coat collagen I coated 96-well plates (ThermoFisher, Cat. 877272) at a density of 50,000 cells/well.
  • Plated cells were allowed to settle and adhere for 24 hours in a tissue culture incubator (37° C. and 5% CO 2 atmosphere) prior to LNP administration. After incubation cells were checked for monolayer formation and media was replaced with hepatocyte culture medium with serum-free supplement pack (Invitroven, Cat. A1217601 and CM4000).
  • LNP formulations for this study were prepared as described above.
  • lipid nanoparticle formulations containing modified sgRNAs were tested on primary cyno hepatocytes to generate a dose response curve.
  • LNPs were incubated in hepatocyte maintenance media containing 6% cyno serum at 37° C. for 5 minutes.
  • Post-incubation the LNPs were added onto the primary cyno hepatocytes in an 8 point 2-fold dose response curve starting at 100 ng mRNA.
  • the cells were lysed 72 hours post treatment for NGS analysis as described in Example 1. Percent editing was determined for various LNP compositions and the data are graphed in FIG. 8 A .
  • the % editing with Cas9 mRNA (SEQ ID NO 48) and U-depleted Cas9 mRNA (SEQ I NO:43) is presented in FIG. 8 B .
  • LNP compositions are described in Table 2 (LNP 897) and Table 5 (LNP 1021, 1022, 1023, 1024, and 1025).
  • RNA Cargo mRNA and gRNA Coformulations
  • LNP formulations prepared from the mRNA described and sg282 (SEQ ID NO: 42; G282) as described in Example 2 with Lipid A, cholesterol, DSPC, and PEG2k-DMG in a 50:38:9:3 molar ratio and with an N:P ratio of 6.
  • the gRNA:Cas9 mRNA weight ratios of the formulations were as shown in Table 29.
  • RNA total RNA
  • animals were sacrificed, blood and the liver were collected, and serum TTR and liver editing were measured as described in Example 1. Serum TTR and liver editing results are shown in FIGS. 9 A and 9 B .
  • Negative control mice were dosed with TSS vehicle.
  • animals were sacrificed, blood and the liver were collected, and serum TTR and liver editing were measured. Serum TTR and liver editing results are shown in FIG. 9 C and FIG. 9 D .
  • Negative control mice were dosed with TSS vehicle.
  • LNP formulations were prepared with the mRNA of Example 2 and sg534 (SEQ ID NO: 72; G534), as described in Example 2.
  • the lipid nanoparticle components were dissolved in 100% ethanol with the lipid component molar ratios set forth below.
  • the RNA cargos were prepared in a buffer of 25 mM citrate and 100 mM NaCl at pH 5.0, resulting in a concentration of RNA cargo of approximately 0.45 mg/mL.
  • the LNPs were formulated with a lipid amine to RNA phosphate (N:P) molar ratio of about 6 with the ratio of gRNA to mRNA at 1:2 by weight.
  • N:P lipid amine to RNA phosphate
  • LNP formulations were analyzed for average particle size, polydispersity (pdi), total RNA content and encapsulation efficiency of RNA as described in Example 1. Analysis of average particle size, polydispersity (PDI), total RNA content and encapsulation efficiency of RNA are shown in Table 30. Molar ratios of lipid are provided as amine lipid (Lipid A)/neutral lipid/helper lipid (cholesterol)/PEG lipid (PEG2k-DMG). The neutral lipid was DSP, DPPC, or absent, as specified.
  • RNA guide RNA and mRNA
  • serum TTR and liver editing were measured as described in Example 1. Negative control animals were dosed with TSS vehicle. Serum TTR and liver editing results are shown in FIGS. 10 A and 10 B , and in Table 30 (above).
  • Transcript sequences generally include GGG as the first three nucleotides for use with ARCA or AGG as the first three nucleotides for use with CleanCapTM. Accordingly, the first three nucleotides can be modified for use with other capping approaches, such as Vaccinia capping enzyme.
  • Promoters and poly-A sequences are not included in the transcript sequences.
  • a promoter such as a T7 promoter (SEQ ID NO: 31) and a poly-A sequence such as SEQ ID NO: 62 or 63 can be appended to the disclosed transcript sequences at the 5′ and 3′ ends, respectively.
  • Most nucleotide sequences are provided as DNA but can be readily converted to RNA by changing Ts to Us.
  • sequence table provides a listing of sequences disclosed herein. It is understood that if a DNA sequence (comprising Ts) is referenced with respect to an RNA, then Ts should be replaced with Us (which may be modified or unmodified depending on the context), and vice versa.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Genetics & Genomics (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Biomedical Technology (AREA)
  • General Health & Medical Sciences (AREA)
  • Organic Chemistry (AREA)
  • Wood Science & Technology (AREA)
  • Zoology (AREA)
  • Molecular Biology (AREA)
  • Biotechnology (AREA)
  • General Engineering & Computer Science (AREA)
  • Medicinal Chemistry (AREA)
  • Animal Behavior & Ethology (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Epidemiology (AREA)
  • Biochemistry (AREA)
  • Microbiology (AREA)
  • Physics & Mathematics (AREA)
  • Biophysics (AREA)
  • Plant Pathology (AREA)
  • Dispersion Chemistry (AREA)
  • Optics & Photonics (AREA)
  • Dermatology (AREA)
  • Nanotechnology (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Medicinal Preparation (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Enzymes And Modification Thereof (AREA)
  • Medicines Containing Material From Animals Or Micro-Organisms (AREA)
US16/651,911 2017-09-29 2018-09-28 Formulations Pending US20230140670A1 (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
US16/651,911 US20230140670A1 (en) 2017-09-29 2018-09-28 Formulations

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
US201762566240P 2017-09-29 2017-09-29
US16/651,911 US20230140670A1 (en) 2017-09-29 2018-09-28 Formulations
PCT/US2018/053559 WO2019067992A1 (en) 2017-09-29 2018-09-28 Formulations

Publications (1)

Publication Number Publication Date
US20230140670A1 true US20230140670A1 (en) 2023-05-04

Family

ID=64267891

Family Applications (1)

Application Number Title Priority Date Filing Date
US16/651,911 Pending US20230140670A1 (en) 2017-09-29 2018-09-28 Formulations

Country Status (30)

Country Link
US (1) US20230140670A1 (https=)
EP (2) EP4385514A3 (https=)
JP (3) JP7284179B2 (https=)
KR (2) KR20250042147A (https=)
CN (1) CN111406108A (https=)
AR (1) AR113031A1 (https=)
AU (2) AU2018338915C1 (https=)
BR (1) BR112020006300A2 (https=)
CA (1) CA3077413A1 (https=)
DK (1) DK3688162T3 (https=)
EA (1) EA202090868A1 (https=)
ES (1) ES2980114T3 (https=)
FI (1) FI3688162T3 (https=)
HR (1) HRP20240627T1 (https=)
HU (1) HUE066630T2 (https=)
IL (2) IL273541B2 (https=)
LT (1) LT3688162T (https=)
MX (2) MX2020007148A (https=)
MY (1) MY204993A (https=)
PH (1) PH12020552306A1 (https=)
PL (1) PL3688162T3 (https=)
PT (1) PT3688162T (https=)
RS (1) RS65493B1 (https=)
SA (1) SA520411636B1 (https=)
SG (1) SG11202002653UA (https=)
SI (1) SI3688162T1 (https=)
SM (1) SMT202400172T1 (https=)
TW (1) TWI833708B (https=)
UA (1) UA129257C2 (https=)
WO (1) WO2019067992A1 (https=)

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2023225572A2 (en) 2022-05-17 2023-11-23 Nvelop Therapeutics, Inc. Compositions and methods for efficient in vivo delivery
US12319938B2 (en) 2020-07-24 2025-06-03 The General Hospital Corporation Enhanced virus-like particles and methods of use thereof for delivery to cells
US12351815B2 (en) 2019-06-13 2025-07-08 The General Hospital Corporation Engineered human-endogenous virus-like particles and methods of use thereof for delivery to cells
WO2026003582A2 (en) 2024-06-27 2026-01-02 Axelyf ehf. Lipids and lipid nanoparticles

Families Citing this family (137)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US9340799B2 (en) 2013-09-06 2016-05-17 President And Fellows Of Harvard College MRNA-sensing switchable gRNAs
US12491261B2 (en) 2016-10-26 2025-12-09 Acuitas Therapeutics, Inc. Lipid nanoparticle formulations
JP2020510038A (ja) 2017-03-09 2020-04-02 プレジデント アンド フェローズ オブ ハーバード カレッジ がんワクチン
EP3592853A1 (en) 2017-03-09 2020-01-15 President and Fellows of Harvard College Suppression of pain by gene editing
WO2019023680A1 (en) 2017-07-28 2019-01-31 President And Fellows Of Harvard College METHODS AND COMPOSITIONS FOR EVOLUTION OF BASIC EDITORS USING PHAGE-ASSISTED CONTINUOUS EVOLUTION (PACE)
US12522807B2 (en) 2018-07-09 2026-01-13 The Broad Institute, Inc. RNA programmable epigenetic RNA modifiers and uses thereof
JP7543259B2 (ja) 2018-10-18 2024-09-02 アクイタス セラピューティクス インコーポレイテッド 活性剤の脂質ナノ粒子送達のための脂質
JP7578590B2 (ja) 2018-10-18 2024-11-06 インテリア セラピューティクス，インコーポレーテッド 第ｉｘ因子を発現するための組成物及び方法
US12351837B2 (en) 2019-01-23 2025-07-08 The Broad Institute, Inc. Supernegatively charged proteins and uses thereof
JP7657726B2 (ja) 2019-03-19 2025-04-07 ザ ブロード インスティテュート，インコーポレーテッド 編集ヌクレオチド配列を編集するための方法および組成物
EP3946598A1 (en) 2019-03-28 2022-02-09 Intellia Therapeutics, Inc. Compositions and methods comprising a ttr guide rna and a polynucleotide encoding an rna-guided dna binding agent
AU2020248337A1 (en) * 2019-03-28 2021-11-04 Intellia Therapeutics, Inc. Compositions and methods for ttr gene editing and treating ATTR amyloidosis comprising a corticosteroid or use thereof
US12473543B2 (en) 2019-04-17 2025-11-18 The Broad Institute, Inc. Adenine base editors with reduced off-target effects
WO2021008708A1 (en) * 2019-07-18 2021-01-21 Biontech Rna Pharmaceuticals Gmbh Method for determining at least one parameter of a sample composition comprising nucleic acid, such as rna, and optionally particles
WO2021016075A1 (en) 2019-07-19 2021-01-28 Flagship Pioneering Innovations Vi, Llc Recombinase compositions and methods of use
US20230097090A1 (en) * 2019-08-14 2023-03-30 Acuitas Therapeutics, Inc. Improved lipid nanoparticles for delivery of nucleic acids
US12521451B2 (en) 2019-11-08 2026-01-13 Regeneron Pharmaceuticals, Inc. CRISPR and AAV strategies for x-linked juvenile retinoschisis therapy
MX2022006950A (es) * 2019-12-11 2022-11-07 Intellia Therapeutics Inc Arn guía modificados para edición de genes.
JP2023512758A (ja) 2020-02-07 2023-03-29 インテリア セラピューティクス，インコーポレイテッド カリクレイン（ｋｌｋｂ１）遺伝子編集のための組成物および方法
IL296024A (en) 2020-03-04 2022-10-01 Flagship Pioneering Innovations Vi Llc Methods and compositions for modulating a genome
GB2632565B (en) 2020-04-09 2025-06-04 Verve Therapeutics Inc Base editing of PCSK9 and methods of using same for treatment of disease
KR20230017783A (ko) 2020-04-28 2023-02-06 인텔리아 테라퓨틱스, 인크. 시험관내 세포 전달 방법
IL298363A (en) 2020-05-20 2023-01-01 Flagship Pioneering Innovations Vi Llc Immunogenic compositions and uses thereof
CA3179420A1 (en) 2020-05-20 2021-11-25 Avak Kahvejian Coronavirus antigen compositions and their uses
CA3180101A1 (en) 2020-05-29 2021-12-02 Flagship Pioneering Innovations Vi, Llc Trem compositions and methods relating thereto
CA3182026A1 (en) 2020-05-29 2021-12-02 Flagship Pioneering Innovations Vi, Llc. Trem compositions and methods relating thereto
MX2023002439A (es) 2020-09-03 2023-05-09 Flagship Pioneering Innovations Vi Llc Composiciones immunogénicas y usos de las mismas.
JP2023549011A (ja) 2020-09-15 2023-11-22 ヴァーヴ・セラピューティクス，インコーポレーテッド 遺伝子編集のための脂質製剤
IL303195A (en) 2020-11-25 2023-07-01 Akagera Medicines Inc Lipid nanoparticles for delivery of nucleic acids and related methods of use
CA3204997A1 (en) 2020-12-11 2022-06-16 Intellia Therapeutics, Inc. Compositions and methods for reducing mhc class ii in a cell
CR20230305A (es) 2020-12-11 2023-11-10 Intellia Therapeutics Inc Polinucleótidos, composiciones y métodos para la edición del genoma que implican desaminación
TW202242101A (zh) 2020-12-23 2022-11-01 美商英特利亞醫療公司 用於細胞中基因修飾ciita之組合物及方法
AU2021409732A1 (en) 2020-12-23 2023-07-20 Intellia Therapeutics, Inc. Compositions and methods for reducing hla-a in a cell
IL303886A (en) 2020-12-23 2023-08-01 Flagship Pioneering Inc Compositions of modified trems and uses thereof
AU2021411521A1 (en) 2020-12-30 2023-08-03 Intellia Therapeutics, Inc. Engineered t cells
WO2022175815A1 (en) * 2021-02-19 2022-08-25 Pfizer Inc. Methods of protecting rna
JP2024512669A (ja) 2021-03-31 2024-03-19 フラグシップ パイオニアリング イノベーションズ ブイ，インコーポレーテッド タノトランスミッションポリペプチド及び癌の処置におけるそれらの使用
CR20230535A (es) 2021-04-17 2024-02-16 Intellia Therapeutics Inc Inhibidores de proteína cinasa dependiente de adn y composiciones y usos de estos
KR20240032013A (ko) 2021-06-10 2024-03-08 인텔리아 테라퓨틱스, 인크. 유전자 편집을 위한 내부 링커를 포함하는 변형된 가이드 rna
KR20240038705A (ko) 2021-06-22 2024-03-25 인텔리아 테라퓨틱스, 인크. 간 유전자의 생체 내 편집 방법
WO2023009547A1 (en) 2021-07-26 2023-02-02 Flagship Pioneering Innovations Vi, Llc Trem compositions and uses thereof
JP2024533311A (ja) 2021-09-08 2024-09-12 フラッグシップ パイオニアリング イノベーションズ シックス，エルエルシー ゲノムを調節するための方法及び組成物
ES2994006T3 (en) 2021-09-17 2025-01-16 Flagship Pioneering Innovations Vi Llc Compositions and methods for producing circular polyribonucleotides
JP2024538144A (ja) 2021-10-18 2024-10-18 フラッグシップ パイオニアリング イノベーションズ シックス，エルエルシー ポリリボヌクレオチドを精製するための組成物及び方法
EP4423271A2 (en) 2021-10-28 2024-09-04 Regeneron Pharmaceuticals, Inc. Crispr/cas-related methods and compositions for knocking out c5
EP4426822A2 (en) 2021-11-03 2024-09-11 Intellia Therapeutics, Inc. Polynucleotides, compositions, and methods for genome editing
EP4436984A1 (en) 2021-11-24 2024-10-02 Flagship Pioneering Innovations VI, LLC Coronavirus immunogen compositions and their uses
AU2022397292A1 (en) 2021-11-24 2024-05-30 Flagship Pioneering Innovations Vi, Llc Varicella-zoster virus immunogen compositions and their uses
MX2024006440A (es) 2021-11-24 2024-08-19 Flagship Pioneering Innovations Vi Llc Composiciones inmunogenicas y sus usos.
US20250223584A2 (en) 2021-12-03 2025-07-10 The Broad Institute, Inc. Compositions and methods for efficient in vivo delivery
KR20240117149A (ko) 2021-12-22 2024-07-31 플래그쉽 파이어니어링 이노베이션스 브이아이, 엘엘씨 폴리리보뉴클레오티드를 정제하기 위한 조성물 및 방법
MX2024007870A (es) 2021-12-23 2024-08-15 Flagship Pioneering Innovations Vi Llc Polirribonucleotidos circulares que codifican polipeptidos antifusogenicos.
EP4473103A2 (en) 2022-02-02 2024-12-11 Regeneron Pharmaceuticals, Inc. Anti-tfr:gaa and anti-cd63:gaa insertions for treatment of pompe disease
AU2023238223A1 (en) 2022-03-23 2024-10-10 Nanovation Therapeutics Inc. High sterol-containing lipid nanoparticles
WO2023185697A2 (en) 2022-03-29 2023-10-05 Accuredit Therapeutics (Suzhou) Co., Ltd. Compositions and methods for treatment of transthyretin amyloidosis
CN119654166A (zh) 2022-04-01 2025-03-18 内诺维什疗法股份有限公司 mRNA递送方法及其组合物
JP2025511756A (ja) 2022-04-08 2025-04-16 フラッグシップ パイオニアリング イノベーションズ セブン，エルエルシー ワクチン及び関連する方法
US20250268940A1 (en) 2022-04-19 2025-08-28 Intellia Therapeutics, Inc. Chimeric antigen receptor compositions and uses
WO2023212677A2 (en) 2022-04-29 2023-11-02 Regeneron Pharmaceuticals, Inc. Identification of tissue-specific extragenic safe harbors for gene therapy approaches
US20250302998A1 (en) 2022-05-09 2025-10-02 Regeneron Pharmaceuticals, Inc. Vectors and methods for in vivo antibody production
US20250295687A1 (en) 2022-05-09 2025-09-25 Flagship Pioneering Innovations Vi, Llc Trem compositions and methods of use for treating proliferative disorders
JP2025516638A (ja) 2022-05-13 2025-05-30 フラッグシップ パイオニアリング イノベーションズ セブン，エルエルシー 二本鎖ｄｎａ組成物及び関連する方法
EP4531819A2 (en) 2022-05-25 2025-04-09 Akagera Medicines, Inc. Lipid nanoparticles for delivery of nucleic acids and methods of use thereof
CN119421951A (zh) 2022-05-31 2025-02-11 瑞泽恩制药公司 用于c9orf72重复序列扩增疾病的crispr干扰疗法
WO2023235725A2 (en) 2022-05-31 2023-12-07 Regeneron Pharmaceuticals, Inc. Crispr-based therapeutics for c9orf72 repeat expansion disease
JP2023181989A (ja) * 2022-06-13 2023-12-25 上海臻上医薬科技有限公司 マイクロニードル注射製剤およびその使用
CN119384498A (zh) 2022-06-16 2025-01-28 因特利亚治疗公司 用于对细胞进行遗传修饰的方法和组合物
US20250179492A1 (en) 2022-06-22 2025-06-05 Flagship Pioneering Innovations Vi, Llc Compositions of modified trems and uses thereof
CN119585419A (zh) 2022-06-29 2025-03-07 因特利亚治疗公司 工程化t细胞
IL318625A (en) 2022-07-29 2025-03-01 Regeneron Pharma Compositions and methods for transferrin receptor (TFR)-mediated delivery to brain and muscle
JP2025525778A (ja) 2022-08-01 2025-08-07 フラッグシップ パイオニアリング イノベーションズ セブン，エルエルシー 免疫調節タンパク質及び関連方法
EP4569112A1 (en) 2022-08-12 2025-06-18 Remix Therapeutics Inc. Methods and compositions for modulating splicing at alternative splice sites
EP4590820A2 (en) 2022-09-22 2025-07-30 Accuredit Therapeutics (Suzhou) Co., Ltd. Compositions and methods for treatment of hypercholesterolemia and/or cardiovascular disease
WO2024077191A1 (en) 2022-10-05 2024-04-11 Flagship Pioneering Innovations V, Inc. Nucleic acid molecules encoding trif and additionalpolypeptides and their use in treating cancer
AU2023372397A1 (en) 2022-10-31 2025-05-08 Flagship Pioneering Innovations Vi, Llc Compositions and methods for purifying polyribonucleotides
IL320016A (en) 2022-11-04 2025-06-01 Regeneron Pharma Calcium voltage-gated channel auxiliary subunit gamma 1 (cacng1) binding proteins and cacng1-mediated delivery to skeletal muscle
EP4615465A1 (en) 2022-11-08 2025-09-17 Flagship Pioneering Innovations VI, LLC Compositions and methods for producing circular polyribonucleotides
EP4615424A1 (en) 2022-11-10 2025-09-17 Sail Biomedicines, Inc. Rna compositions comprising lipid nanoparticles or lipid reconstructed natural messenger packs
AU2023379457A1 (en) 2022-11-14 2025-05-15 Regeneron Pharmaceuticals, Inc. Compositions and methods for fibroblast growth factor receptor 3-mediated delivery to astrocytes
TW202430215A (zh) 2022-12-14 2024-08-01 美商旗艦先鋒創新有限責任（Ｖｉｉ）公司 用於將治療劑遞送至骨之組成物和方法
CN118236344A (zh) * 2022-12-15 2024-06-25 深圳瑞吉生物科技有限公司 一种核酸脂质纳米载体及其制备方法与应用
EP4637787A2 (en) * 2022-12-20 2025-10-29 Seqirus Inc. Lipid nanoparticle composition
WO2024138189A2 (en) 2022-12-22 2024-06-27 Intellia Therapeutics, Inc. Methods for analyzing nucleic acid cargos of lipid nucleic acid assemblies
WO2024138115A1 (en) 2022-12-23 2024-06-27 Intellia Theraperutics, Inc. Systems and methods for genomic editing
US20240252520A1 (en) 2023-01-09 2024-08-01 Beth Israel Deaconess Medical Center, Inc. Therapeutic agents and their use for treating chronic wounds
WO2024151583A2 (en) 2023-01-09 2024-07-18 Flagship Pioneering Innovations Vii, Llc Vaccines and related methods
WO2024151687A1 (en) 2023-01-09 2024-07-18 Flagship Pioneering Innovations V, Inc. Genetic switches and their use in treating cancer
US20240238473A1 (en) 2023-01-09 2024-07-18 Beth Israel Deaconess Medical Center, Inc. Recombinant nucleic acid molecules and their use in wound healing
WO2024167885A1 (en) 2023-02-06 2024-08-15 Flagship Pioneering Innovations Vii, Llc Immunomodulatory compositions and related methods
IL322468A (en) 2023-02-13 2025-09-01 Flagship Pioneering Innovations Vii Llc Ionizable lipids containing degradable crosslinkers and lipid carriers for therapeutic compositions
IL322378A (en) 2023-02-17 2025-09-01 Flagship Pioneering Innovations Vii Llc DNA compositions containing modified cytosine
JP2026507513A (ja) 2023-02-17 2026-03-04 フラッグシップ パイオニアリング イノベーションズ セブン，エルエルシー 修飾されたウラシルを含むｄｎａ組成物
EP4680276A1 (en) 2023-03-15 2026-01-21 Flagship Pioneering Innovations VI, LLC Immunogenic compositions and uses thereof
WO2024192420A1 (en) 2023-03-15 2024-09-19 Flagship Pioneering Innovations Vi, Llc Compositions comprising polyribonucleotides and uses thereof
WO2024216191A1 (en) 2023-04-12 2024-10-17 Flagship Pioneering Innovations Vi, Llc Modified trems, compositions, and related methods thereof
WO2024216128A1 (en) 2023-04-12 2024-10-17 Flagship Pioneering Innovations Vi, Llc Trems for use in correction of missense mutations
WO2024220746A2 (en) 2023-04-21 2024-10-24 Flagship Pioneering Innovations Vii, Llc Rnai agents targeting fatty acid synthase and related methods
WO2024258829A1 (en) 2023-06-12 2024-12-19 Flagship Pioneering Innovations Vii, Llc Sars-cov-2 vaccine compositions and related methods
WO2024259135A1 (en) 2023-06-13 2024-12-19 Intellia Therapeutics, Inc. Assays for analysis of ribonucleic acid (rna) molecules
WO2025006684A1 (en) 2023-06-28 2025-01-02 Flagship Pioneering Innovations Vi, Llc Circular polyribonucleotides encoding antifusogenic polypeptides
AU2024309884A1 (en) 2023-06-30 2025-12-18 Regeneron Pharmaceuticals, Inc. Methods and compositions for increasing homology-directed repair
AU2024299627A1 (en) 2023-07-25 2026-01-22 Flagship Pioneering Innovations Vii, Llc Cas endonucleases and related methods
US20250049896A1 (en) 2023-07-28 2025-02-13 Regeneron Pharmaceuticals, Inc. Anti-tfr:acid sphingomyelinase for treatment of acid sphingomyelinase deficiency
AU2024315073A1 (en) 2023-07-28 2026-01-22 Regeneron Pharmaceuticals, Inc. Use of bgh-sv40l tandem polya to enhance transgene expression during unidirectional gene insertion
AU2024324870A1 (en) 2023-08-14 2026-02-12 Intellia Therapeutics, Inc. Compositions and methods for genetically modifying transforming growth factor beta receptor type 2 (tgfβr2)
TW202521564A (zh) 2023-08-14 2025-06-01 美商英特利亞醫療公司 用於基於細胞之療法的ｃｄ７０ ｃａｒ－ｔ組合物及方法
WO2025038637A1 (en) 2023-08-14 2025-02-20 Intellia Therapeutics, Inc. Compositions and methods for genetically modifying transforming growth factor beta receptor type 2 (tgfβr2)
TW202515994A (zh) 2023-08-14 2025-04-16 美商英特利亞醫療公司 用於對ｃｄ７０進行基因修飾之組合物及方法
WO2025042786A1 (en) 2023-08-18 2025-02-27 Flagship Pioneering Innovations Vi, Llc Compositions comprising circular polyribonucleotides and uses thereof
WO2025049481A1 (en) 2023-08-28 2025-03-06 Intellia Therapeutics, Inc. Methods of editing an hla-a gene in vitro
CN121752723A (zh) * 2023-08-31 2026-03-27 正基基因科技有限公司 编码crispr相关蛋白的核酸及其应用
WO2025054236A2 (en) 2023-09-06 2025-03-13 Flagship Pioneering Innovations Vii, Llc Sars-cov-2 vaccine compositions and related methods
WO2025064401A1 (en) 2023-09-18 2025-03-27 Intellia Therapeutics, Inc. Nuclease resistant single stranded dna product for non-viral delivery to a cell and methods of production thereof
AU2024344058A1 (en) 2023-09-18 2026-04-02 Flagship Pioneering Innovations Vii, Llc Ionizable lipidoid compositions and therapeutic uses thereof
WO2025064396A1 (en) 2023-09-18 2025-03-27 Intellia Therapeutics, Inc. Nuclease resistant double stranded dna product for non-viral delivery to a cell and methods of production thereof
WO2025072331A1 (en) 2023-09-26 2025-04-03 Flagship Pioneering Innovations Vii, Llc Cas nucleases and related methods
WO2025096807A2 (en) 2023-10-31 2025-05-08 Flagship Pioneering Innovations Vii, Llc Novel therapeutic dna forms
WO2025106670A1 (en) 2023-11-14 2025-05-22 Flagship Pioneering Innovations Vii, Llc Ionizable lipidoid compositions and therapeutic uses thereof
US20250161347A1 (en) 2023-11-22 2025-05-22 Flagship Pioneering Innovations Vii, Llc Methods and compositions for treating non-alcoholic fatty liver disease
WO2025117877A2 (en) 2023-12-01 2025-06-05 Flagship Pioneering Innovations Vii, Llc Cas nucleases and related methods
WO2025128871A2 (en) 2023-12-13 2025-06-19 Renagade Therapeutics Management Inc. Lipid nanoparticles comprising coding rna molecules for use in gene editing and as vaccines and therapeutic agents
WO2025137301A1 (en) 2023-12-20 2025-06-26 Intellia Therapeutics, Inc. Methods for rapid engineering of cells
WO2025137439A2 (en) 2023-12-20 2025-06-26 Intellia Therapeutics, Inc. Engineered t cells
TW202545974A (zh) 2024-01-26 2025-12-01 美商旗艦先鋒創新有限責任（Ｖｉｉ）公司 免疫受體抑制蛋白及相關方法
WO2025160340A2 (en) 2024-01-26 2025-07-31 Regeneron Pharmaceuticals, Inc. Combination immunosuppression for inhibiting an immune response and enabling immunogen administration and re-administration
WO2025194019A1 (en) 2024-03-14 2025-09-18 Flagship Pioneering Innovations Vii, Llc Methods for treating liver fibrosis and non-alcoholic fatty liver disease
WO2025217275A2 (en) 2024-04-10 2025-10-16 Flagship Pioneering Innovations Vii, Llc Immune cell targeted compositions and related methods
WO2025240680A1 (en) 2024-05-16 2025-11-20 Flagship Pioneering Innovations Vii, Llc Immunoreceptor inhibitory proteins and related methods
WO2025240946A1 (en) 2024-05-17 2025-11-20 Intellia Therapeutics, Inc. Lipid nanoparticles and lipid nanoparticle compositions
WO2025245188A2 (en) 2024-05-21 2025-11-27 Flagship Pioneering Innovations Vii, Llc Methods of treating liver steatosis and non-alcoholic fatty liver disease
WO2025245111A1 (en) 2024-05-22 2025-11-27 Flagship Pioneering Innovations Vii, Llc Immunoreceptor targeting proteins and related methods
WO2025255308A1 (en) 2024-06-07 2025-12-11 Intellia Therapeutics, Inc. Cd8 co-receptor chimeric polypeptides in tcr cell therapy
WO2025265017A1 (en) 2024-06-20 2025-12-26 Regeneron Pharmaceuticals, Inc. Ass1 gene insertion for the treatment of citrullinemia type i
US20260000702A1 (en) 2024-06-26 2026-01-01 Flagship Pioneering Innovations Vii, Llc Therapeutic circular dna forms
WO2026055547A1 (en) 2024-09-06 2026-03-12 Flagship Pioneering Innovations Vii, Llc Dna compositions and related methods
WO2026055543A1 (en) 2024-09-06 2026-03-12 Flagship Pioneering Innovations Vii, Llc Modified dna compositions and related methods
WO2026064313A1 (en) 2024-09-17 2026-03-26 Flagship Pioneering Innovations Vii, Llc Rna compositions and related methods

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20160311759A1 (en) * 2013-12-19 2016-10-27 Luis Brito Lipids and Lipid Compositions for the Delivery of Active Agents
WO2017127750A1 (en) * 2016-01-22 2017-07-27 Modernatx, Inc. Messenger ribonucleic acids for the production of intracellular binding polypeptides and methods of use thereof

Family Cites Families (19)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2011076807A2 (en) 2009-12-23 2011-06-30 Novartis Ag Lipids, lipid compositions, and methods of using them
US8691750B2 (en) * 2011-05-17 2014-04-08 Axolabs Gmbh Lipids and compositions for intracellular delivery of biologically active compounds
WO2014093694A1 (en) 2012-12-12 2014-06-19 The Broad Institute, Inc. Crispr-cas nickase systems, methods and compositions for sequence manipulation in eukaryotes
DK2931898T3 (en) 2012-12-12 2016-06-20 Massachusetts Inst Technology CONSTRUCTION AND OPTIMIZATION OF SYSTEMS, PROCEDURES AND COMPOSITIONS FOR SEQUENCE MANIPULATION WITH FUNCTIONAL DOMAINS
CA2895155C (en) 2012-12-17 2021-07-06 President And Fellows Of Harvard College Rna-guided human genome engineering
EP3608308B1 (en) 2013-03-08 2021-07-21 Novartis AG Lipids and lipid compositions for the delivery of active agents
CA2906732C (en) * 2013-03-15 2023-08-08 The University Of British Columbia Lipid nanoparticles for transfection and related methods
SMT202100691T1 (it) * 2013-07-11 2022-01-10 Modernatx Inc Composizioni 5 comprendenti polinucleotidi sintetici che codificano proteine correlate a crispr e sgrna sintetici e metodi d'uso
EP3470089A1 (en) * 2013-12-12 2019-04-17 The Broad Institute Inc. Delivery, use and therapeutic applications of the crispr-cas systems and compositions for targeting disorders and diseases using particle delivery components
US20150165054A1 (en) 2013-12-12 2015-06-18 President And Fellows Of Harvard College Methods for correcting caspase-9 point mutations
EP3169309B1 (en) 2014-07-16 2023-05-10 Novartis AG Method of encapsulating a nucleic acid in a lipid nanoparticle host
EP3265559B1 (en) 2015-03-03 2021-01-06 The General Hospital Corporation Engineered crispr-cas9 nucleases with altered pam specificity
AU2016237148A1 (en) * 2015-03-24 2017-11-23 Kyowa Hakko Kirin Co., Ltd. Nucleic acid-containing lipid nanoparticles
US10626393B2 (en) * 2015-06-04 2020-04-21 Arbutus Biopharma Corporation Delivering CRISPR therapeutics with lipid nanoparticles
US20190054112A1 (en) * 2015-09-18 2019-02-21 Moderna Therapeutics, Inc. Polynucleotide formulations for use in the treatment of renal diseases
CA3006618A1 (en) * 2015-12-01 2017-06-08 Crispr Therapeutics Ag Materials and methods for treatment of alpha-1 antitrypsin deficiency
BR112018012894A2 (en) * 2015-12-23 2018-12-04 Crispr Therapeutics Ag Materials and Methods for Treatment of Amyotrophic Lateral Sclerosis and / or Frontotemporal Lobular Degeneration
TWI773666B (zh) * 2016-03-30 2022-08-11 美商英特利亞醫療公司 Crispr/cas 組分之脂質奈米粒子調配物
AU2017379073B2 (en) * 2016-12-22 2023-12-14 Intellia Therapeutics, Inc. Compositions and methods for treating alpha-1 antitrypsin deficiency

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20160311759A1 (en) * 2013-12-19 2016-10-27 Luis Brito Lipids and Lipid Compositions for the Delivery of Active Agents
WO2017127750A1 (en) * 2016-01-22 2017-07-27 Modernatx, Inc. Messenger ribonucleic acids for the production of intracellular binding polypeptides and methods of use thereof

Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US12351815B2 (en) 2019-06-13 2025-07-08 The General Hospital Corporation Engineered human-endogenous virus-like particles and methods of use thereof for delivery to cells
US12351814B2 (en) 2019-06-13 2025-07-08 The General Hospital Corporation Engineered human-endogenous virus-like particles and methods of use thereof for delivery to cells
US12404525B2 (en) 2019-06-13 2025-09-02 The General Hospital Corporation Engineered human-endogenous virus-like particles and methods of use thereof for delivery to cells
US12319938B2 (en) 2020-07-24 2025-06-03 The General Hospital Corporation Enhanced virus-like particles and methods of use thereof for delivery to cells
WO2023225572A2 (en) 2022-05-17 2023-11-23 Nvelop Therapeutics, Inc. Compositions and methods for efficient in vivo delivery
WO2026003582A2 (en) 2024-06-27 2026-01-02 Axelyf ehf. Lipids and lipid nanoparticles

Also Published As

Publication number Publication date
SG11202002653UA (en) 2020-04-29
DK3688162T3 (da) 2024-05-13
SI3688162T1 (sl) 2024-06-28
FI3688162T3 (fi) 2024-05-15
SMT202400172T1 (it) 2024-07-09
SA520411636B1 (ar) 2024-06-09
RS65493B1 (sr) 2024-05-31
MX2024013597A (es) 2024-12-06
ES2980114T3 (es) 2024-09-30
IL273541B1 (en) 2025-06-01
JP2020536125A (ja) 2020-12-10
EP3688162B1 (en) 2024-03-06
IL273541B2 (en) 2025-10-01
KR20200079497A (ko) 2020-07-03
MX2020007148A (es) 2020-10-08
IL273541A (en) 2020-05-31
CN111406108A (zh) 2020-07-10
KR20250042147A (ko) 2025-03-26
UA129257C2 (uk) 2025-03-05
HUE066630T2 (hu) 2024-08-28
EP3688162A1 (en) 2020-08-05
EP4385514A2 (en) 2024-06-19
AU2018338915A1 (en) 2020-04-23
AR113031A1 (es) 2020-01-15
HRP20240627T1 (hr) 2024-08-02
BR112020006300A2 (pt) 2020-10-20
PT3688162T (pt) 2024-04-23
JP2023103421A (ja) 2023-07-26
AU2018338915B2 (en) 2024-10-24
EA202090868A1 (ru) 2020-09-16
TW201924724A (zh) 2019-07-01
JP2025084955A (ja) 2025-06-03
AU2024224004A1 (en) 2024-10-24
JP7284179B2 (ja) 2023-05-30
PH12020552306A1 (en) 2023-07-17
TWI833708B (zh) 2024-03-01
EP4385514A3 (en) 2024-10-23
IL320823A (en) 2025-07-01
WO2019067992A1 (en) 2019-04-04
LT3688162T (lt) 2024-05-27
AU2018338915C1 (en) 2025-05-08
JP7645929B2 (ja) 2025-03-14
CA3077413A1 (en) 2019-04-04
PL3688162T3 (pl) 2024-06-10
MY204993A (en) 2024-09-26

Similar Documents

Publication Publication Date Title
EP3688162B1 (en) Formulations
US20230203480A1 (en) Lipid nanoparticle formulations for crispr/cas components
EP3688173B1 (en) In vitro method of mrna delivery using lipid nanoparticles
US20240124897A1 (en) Compositions and Methods Comprising a TTR Guide RNA and a Polynucleotide Encoding an RNA-Guided DNA Binding Agent
US20230012687A1 (en) Polynucleotides, Compositions, and Methods for Polypeptide Expression
HK40112168A (en) Formulations
HK40025999B (en) Formulations
HK40025999A (en) Formulations
EA047139B1 (ru) Составы
HK40129152A (en) Lipid nanoparticle formulations for crispr/cas components
HK40033101A (en) Formulations
HK40025136B (en) In vitro method of mrna delivery using lipid nanoparticles
HK40025136A (en) In vitro method of mrna delivery using lipid nanoparticles
HK40067192A (zh) 用於多肽表达的多核苷酸、组合物和方法
HK40003257B (en) Lipid nanoparticle formulations for crispr/cas components
HK40003257A (en) Lipid nanoparticle formulations for crispr/cas components

Legal Events

Date Code Title Description
AS Assignment

Owner name: INTELLIA THERAPEUTICS, INC., MASSACHUSETTS

Free format text: ASSIGNMENT OF ASSIGNORS INTEREST;ASSIGNORS:WOOD, KRISTY M.;GARDNER, NOAH P.;SHAH, RUCHI R.;AND OTHERS;REEL/FRAME:053973/0976

Effective date: 20180927

STPP Information on status: patent application and granting procedure in general

Free format text: DOCKETED NEW CASE - READY FOR EXAMINATION

STPP Information on status: patent application and granting procedure in general

Free format text: NON FINAL ACTION MAILED

STPP Information on status: patent application and granting procedure in general

Free format text: RESPONSE TO NON-FINAL OFFICE ACTION ENTERED AND FORWARDED TO EXAMINER

STPP Information on status: patent application and granting procedure in general

Free format text: NON FINAL ACTION MAILED

STPP Information on status: patent application and granting procedure in general

Free format text: RESPONSE TO NON-FINAL OFFICE ACTION ENTERED AND FORWARDED TO EXAMINER

STPP Information on status: patent application and granting procedure in general

Free format text: NON FINAL ACTION MAILED

STPP Information on status: patent application and granting procedure in general

Free format text: RESPONSE AFTER FINAL ACTION FORWARDED TO EXAMINER

STPP Information on status: patent application and granting procedure in general

Free format text: ADVISORY ACTION COUNTED, NOT YET MAILED

STPP Information on status: patent application and granting procedure in general

Free format text: ADVISORY ACTION MAILED

STPP Information on status: patent application and granting procedure in general

Free format text: DOCKETED NEW CASE - READY FOR EXAMINATION

STPP Information on status: patent application and granting procedure in general

Free format text: NON FINAL ACTION COUNTED, NOT YET MAILED

STPP Information on status: patent application and granting procedure in general

Free format text: NON FINAL ACTION MAILED

STPP Information on status: patent application and granting procedure in general

Free format text: NON FINAL ACTION MAILED

STPP Information on status: patent application and granting procedure in general

Free format text: RESPONSE TO NON-FINAL OFFICE ACTION ENTERED AND FORWARDED TO EXAMINER